Stomach Proteases Research Articles

Comparative bioinformatic analysis and biochemical characterization of digestive proteinases from Pacific whiting (Merluccius productus)

The hake fishery plays a crucial role due to its significant economic impact. The genus Merluccius includes 12 extant species found along the coasts of the Americas, Europe, and Africa. However, research on their digestive physiology and the enzymes involved in digestion, including proteases, remains limited. Proteases play a key role in protein digestion, a vital process for all living organisms. This study focused on screening the genomes of eight Merluccius spp. for eight specific proteases previously identified in Merluccius polli. Additionally, the study conducted biochemical analyses of proteases found in the stomach and intestine of Pacific whiting fish (Merluccius productus), comparing the results with the genomic findings. The analysis revealed that proteases across Merluccius spp. are conserved, although with slight variations, particularly in chymotrypsin and aspartic proteases. Biochemical characterization of M. productus identified at least three main proteases in the stomach, active at acidic pH, and at least seven proteases in the intestine, active at alkaline pH, as determined by electrophoresis. Further investigation, including specific inhibition studies, determination of molecular mass, and assessment of pH and temperature preferences for catalysis, revealed that one of the stomach proteases functioning at acidic pH likely belongs to the acid peptidase class, likely pepsin. Similarly, analysis of proteases active at alkaline pH indicated the presence of a chymotrypsin and a trypsin, consistent with genomic findings in M. productus. These results are important as they provide insights into the digestive physiology of Merluccius spp., contributing to a better understanding of their nutritional needs.

Partial characterization of digestive proteases in Pacific red snapper Lutjanus peru Nichols & Murphy, 1922 (Perciformes: Lutjanidae)

Pacific red snapper (Lutjanus peru) is an important commercial species in Mexico with great aquaculture potential; however, digestive physiology is still unknown. Therefore, the objective of the present work was to characterize the digestive proteases of L. peru juvenile using biochemical and electrophoretic techniques. Results showed a higher acid protease activity than the alkaline proteases, trypsin, chymotrypsin, and leucine aminopeptidase (LAP). The optimum temperature for acid proteases was between 30 to 40°C. Trypsin activity showed two maximum peaks of temperature (30 and 50°C), while alkaline proteases, chymotrypsin, and LAP had optimum temperatures of 50, 50 to 60, and 40°C, respectively. Moreover, the optimum pH of acid proteases was between 2 and 3. Also, alkaline proteases, trypsin, chymotrypsin showed pH optimums at pH 6, 9, and 5, respectively, although LAP showed two optimum pH values at 6 and 9. Acid protease zymogram showed three isoforms, totally inhibited by pepstatin A. Alkaline protease zymogram revealed six bands (125.4, 67.2, 57.9, 48.6, 29.8, and 26.9 kDa), which were inhibited by specific serine-proteases and metalloproteases inhibitors. In conclusion, the main digestion in L. peru depends on stomach proteases, which are characteristic of carnivorous fish, followed by intestinal digestion supported mainly by chymotrypsin.

Substitution of fish meal with enzyme‐treated soybean in diets for juvenile largemouth bass ( Micropterus salmoides )

A 63-day feeding trial was conducted to investigate the effects of enzyme-treated soybean (ESB) replacing fish meal (FM) on growth performance, muscle composition, biochemical indices, digestive enzymes activities and intestinal morphology of juvenile largemouth bass (Micropterus salmoides). Largemouth bass with initial weight of (18.14 ± 0.11) g were randomly divided into 5 groups with 3 replicates, each group of 30 fish per replicate. Five diets were formulated by replacing 0, 80, 120, 160 and 200 g kg−1 FM with ESB (ESB0, ESB80, ESB120, ESB160 and ESB200, respectively). Results showed that weight gain rate and specific growth rate significantly improved in ESB160 group (p < .05). Crude protein content in muscle significantly increased with FM replacement amount up to 160 g kg−1, while crude lipid content significantly decreased (p < .05). Activities of lysozyme and catalase reached the highest level in ESB120 group (p < .05). The activity of superoxide dismutase was significantly increased in ESB200 group, while malondialdehyde concentration significantly decreased. With increasing dietary ESB level, activities of protease and lipase in stomach had significant increase in ESB160 group (p < .05). The intestinal wall thickness and villi length in ESB160 group were significantly higher than those in other groups (p < .05). To sum up, the present study suggested that the optimal replacement level of dietary FM by ESB was 160 g kg−1, which means the supplemented levels of FM and ESB in diet were 400 and 260 g kg−1, respectively.

Comparative characterization of digestive proteases in redhead cichlid (Vieja melanurus) and twoband cichlid (Vieja bifasciata) (Percoidei: Cichlidae)

ABSTRACT In the Southeast of Mexico, there are many native cichlids with commercial interest such as redhead cichlid (Vieja melanurus) and twoband cichlid (V. bifasciata), which have a great local demand and excellent meat quality. However, it is necessary to implement their culture based on nutrition studies and digestive biochemistry. This study’s objective was to characterize these two cichlids’ digestive proteases (pH, temperature, and inhibitors) through biochemistry techniques. Results showed that V. melanurus and V. bifasciata have a digestive capacity analogous to other omnivore fishes, where the optimal pH values of stomach proteases (4 and 2, respectively) and intestinal proteases (6 and 12, respectively), the optimal temperature of acid (35°C and 55°C, respectively) and alkaline proteases (45°C and 55°C, respectively) are quite similar. Both species presented high thermal and pH stabilities. Inhibition showed that V. melanurus is more sensitive to specific inhibitors for alkaline proteases than V. bifasciata. In conclusion, V. bisfasciata and V. melanurus have different digestive protease patterns. Both species can hydrolyze different protein ingredients to formulate a specific diet. Nevertheless, V. bifasciata is more resistant to the presence of inhibitors, which allow it to include vegetable proteins in its diet.

The positive effects of single or conjoint administration of lactic acid bacteria on Channa argus: Digestive enzyme activity, antioxidant capacity, intestinal microbiota and morphology

In the present study, we aimed to evaluate the single or conjoint effects of Lactococcus lactis (L. lactis) L19 and Enterococcus faecalis (E. faecalis) W24 isolated from the intestine of Channa argus (C. argus) on digestive enzyme activity, antioxidant capacity, intestinal microbiota and morphology of C. argus (initial weight, 9.50 ± 0.03 g). The fish were fed for 56 days with a basal diet (CK) or diets supplemented with 1.0 × 108 CFU/g of L. lactis (L19), E. faecalis (W24), L. lactis + E. faecalis (L + W). Results indicated that these three lactic acid bacteria (LAB) supplementary diets especially the L19 produced significant improvement in the digestive enzyme activity (protease, amylase and lipase) in liver, stomach and intestine compared with control (P < .05). LAB supplementations were enhanced the liver and intestine antioxidant status (SOD, CAT, GSH-Px, T-AOC and MDA) in comparison to the control (P < .05). Serum biochemical parameters (ALT and AST) of fish fed with LAB supplemented diets showed significantly decreased compared with control (P < .05). The intestinal morphology showed protection in fish fed both L19 or/and W24. High-throughput sequencing revealed L19 supplementation can effectively increase the generation of probiotic bacteria and decrease the abundance of aquatic pathogens. Therefore, supplementation of L. lactis L19 showed more effective protection than E. faecalis W24 or the mixture of the two for promoting digestive enzyme activity, antioxidant capacity, intestinal microbiota and morphology of C. argus.

Partial characterization of digestive proteases in juveniles of Microphis brachyurus (short-tailed pipefish) (Syngnathiformes: Syngnathidae)

ABSTRACT Short-tailed pipe fish (Microphis brachyurus) is a freshwater organism with high economic potential for the aquarium hobby, so it is necessary to implement methods to promote its culture through studies of digestive physiology. General activities of acid and alkaline proteases were evaluated, as well as the effect of pH, temperature and inhibitors. The optimal pH of stomach proteases was 2, while the optimal pH of intestinal proteases was 10. Optimal temperature for the acidic proteases was 35 ºC, while for alkaline proteases it was 45 ºC. Thermal stability showed high resistance at 35 ºC for both acid and alkaline proteases (above 100% residual activity). Acid proteases are resistant at pH 2 (50% of residual activity), meanwhile alkaline proteases were highly resistant at pH 10 (90% of residual activity). Acid proteases were inhibited by 80% with pepstatin A and alkaline proteases were inhibited with TLCK and TPCK for trypsin (75%) and chymotrypsin (80%), respectively. Finally, metallo-proteases were 75% partially inhibited some serine proteases by 75% with EDTA. In conclusion, M. brachyurus has a good digestive capacity, since they can degrade a wide variety of proteins due to their greater proteolytic activity.

Multi spectroscopy and molecular modeling aspects related to drug interaction of aspirin and warfarin with pepsin; structural change and protease activity.

This study evaluates the biochemical interactions between two widely used anticoagulants agents, Aspirin and Warfarin, with the Pepsin as the main stomach protease. These two drugs usually prescribe orally for long period daily use to reduce cardiovascular and thrombi death which leads to being in close contact with Pepsin. This interaction could induce related gastrointestinal problems such as peptic ulcer. In this regard, the conformational changes and enzymatic activity of the Pepsin induced by Aspirin and Warfarin were studied by using several spectroscopic methods along with molecular modeling approaches. Results confirm the formation of stable complexes between protein and drugs which leads to slight subsequent conformational changes of protein structure. The quenching mechanisms for both drug-Pepsin interactions are static. In the case of Warfarin, the hydrophobic interactions are the most important interactions. Also for Aspirin, hydrogen bond and van der Waals forces are mainly involved in the binding process. The Warfarin shows the induction of some conformational changes resulted in suppressing the protease activity and the Aspirin reversely enhanced the enzyme activity function. This study provides useful information regarding the effects of Warfarin and Aspirin on Pepsin which are helpful for the choosing of therapeutics depending on the patients' condition.

Effects of dietary lipid levels on sub-adult triploid rainbow trout (Oncorhynchus mykiss): 1. Growth performance, digestive ability, health status and expression of growth-related genes

A growth trial was conducted to evaluate the effects of dietary lipid levels on the growth performance, feed utilization, digestive ability, health status and expression of growth-related genes of female triploid rainbow trout (Oncorhynchus mykiss). Six isonitrogenous diets, containing 46% protein were formulated with graded lipid levels of 6.6 (Diet 1), 12.3 (Diet 2), 14.8 (Diet 3), 19.5 (Diet 4), 22.8 (Diet 5) and 29.4% (Diet 6). Each diet was fed to quadruplicated groups of fish with initial average weight of 233 g in freshwater cages for 80 days. Results showed that there were significant linear and quadratic responses in growth performance, feed utilization, digestive ability and health status of sub-adult fish (P < .05). Final weight, specific growth rate (SGR), feed intake (FI), stomach protease activity, activity of lipase in pyloric caeca, activity of amylase in pyloric caeca and intestine showed a general increasing trend with increasing dietary protein level. Feed conversion ratio (FCR) decreased with increasing dietary protein level. No significant difference of intestinal morphology was observed in any of the groups (P > .05) except that the highest value for muscular layer thickness and density of goblet cells were found in Diet 6 treatment (P < .05). When examining health status, no negative effects were found in the antioxidative capacity of the intestine and liver (P > .05), however, the lowest value of plasma total anti-oxidative capacity (T-AOC) content and highest plasma malonaldehyde (MDA) content were shown in Diet 6 treatment (P < .05). There was no significant difference in plasma enzymes in any of the groups (P > .05) except for activities of aspartate aminotransferase (AST) and creatine kinase (CK), which increased as dietary lipid levels increased (P < .05). The expression of growth-related genes was at first significantly improved (P < .05) and then kept consistent (P > .05). Broken-line regression analysis of FCR showed that the minimum dietary lipid level of triploid rainbow trout was 23.3% in the present study. In summary, we found that triploid rainbow trout could use or tolerate high dietary lipid level (up to 29.4%) with no negative effect on fish growth, feed utilization, liver or intestine health. The minimum dietary lipid level for sub-adult triploid rainbow trout was estimated to be 23.3% based on FCR under the present conditions.

Partial characterization of digestive proteases in Totoaba (Totoaba macdonaldi)

ABSTRACTThe objective of this study was to characterize the digestive proteases of totoaba (Totoaba macdonaldi). Fish were sacrificed to obtain the multienzymatic extracts from the stomach and intestine, and determine the stability and optimum pH and temperature values. Residual activity and number of isoforms were determined with some inhibitors. Optimal pH of stomach proteases was 2, with stability above 100% at that same pH. Optimum pH of intestinal proteases was between 9 and 11, with stability above 100% between 8-12. Optimum temperature for stomach proteases was 35°C and remained highly stable, while optimum temperature for intestinal proteases was 45°C, with high stability between 35-55°C. Pepstatin A totally inhibited acid protease activity and revealed a single band. SDS-PAGE zymogram revealed 8 bands in the intestine, where phenanthroline inhibited 80% of the total activity. The digestive capacity of T. macdonaldi is characteristic of a strict carnivore, similar to other marine fish species.

Partial characterization of digestive proteases in sheepshead, Archosargus probatocephalus (Spariformes: Sparidae)

ABSTRACT Digestive proteases were partially characterized in sheepshead juveniles, using biochemical and electrophoretic techniques. Results showed higher activity level of the stomach proteases (2.39 ± 0.02 U mg protein-1) compared to the intestinal proteases (1.6 ± 0.1 U mg protein-1). The activity of trypsin, chymotrypsin, leucine aminopeptidase and carboxypeptidase A was also recorded. The optimum temperature of the stomach proteases was recorded at 45 °C, while for intestinal proteases was recorded at 55 °C. Stomach proteases showed less stability to temperature changes than intestinal proteases. An optimum pH of 2 was recorded for stomach proteases with high stability under acidic conditions, while an optimum pH of 9 was recorded for intestinal proteases showing high stability under alkaline conditions. Stomach proteases were inhibited around 78% with Pepstatin A, indicating the presence of pepsin as the main protease. The stomach proteases zymogam revealed one active band with Rf of 0.49, this enzyme was completely inhibited by Pepstatin A. The intestinal proteases zymogram revealed four active proteases (51.3, 34.9, 27.8 and 21.2 kDa) that were inhibited by TLCK, which mainly represent a trypsin-like serine proteases. It can be conclude that digestion in sheepshead can be considered as a carnivorous species with an omnivorous tendency.

Identification of Novel Cryptic Multifunctional Antimicrobial Peptides from the Human Stomach Enabled by a Computational–Experimental Platform

Novel approaches are needed to combat antibiotic resistance. Here, we describe a computational-experimental framework for the discovery of novel cryptic antimicrobial peptides (AMPs). The computational platform, based on previously validated antimicrobial scoring functions, indicated the activation peptide of pepsin A, the main human stomach protease, and its N- and C-terminal halves as antimicrobial peptides. The three peptides from pepsinogen A3 isoform were prepared in a recombinant form using a fusion carrier specifically developed to express toxic peptides in Escherichia coli. Recombinant pepsinogen A3-derived peptides proved to be wide-spectrum antimicrobial agents with MIC values in the range 1.56-50 μM (1.56-12.5 μM for the whole activation peptide). Moreover, the activation peptide was bactericidal at pH 3.5 for relevant foodborne pathogens, suggesting that this new class of previously unexplored AMPs may contribute to microbial surveillance within the human stomach. The peptides showed no toxicity toward human cells and exhibited anti-infective activity in vivo, reducing by up to 4 orders of magnitude the bacterial load in a mouse skin infection model. These peptides thus represent a promising new class of antibiotics. We envision that computationally guided data mining approaches such as the one described here will lead to the discovery of antibiotics from previously unexplored sources.

Evaluation of efficiency of using the enzyme preparation «Celloviridin Dx20» in the content of pig feed

One of the main problems in the feeding of farm animals is the low degree of digestion and assimilation of nutrients in the feed used in animal production. In the bodies of animals such as pigs and poultry enzymes and microflora which are necessary for breaking down cellulose and other polysaccharides of vegetable raw materials are absent, therefore they not only fail to be digested in the gastrointestinal tract, but also obstruct access of other digestive enzymes to the other feed components, in particular intracellular proteins. The only way to solve this problem is deliberate introduction of enzymes into food. Depending on the type and composition of the feed, amylolytic, proteolytic, cellulolytic enzyme preparations are used. The purpose of this work is to assess the effect of the enzyme preparation "Celloviridin Dx20" as a fodder component on the growth and biochemical parameters of young pigs. For this purpose, a scientific and economic experiment on feeding young pigs was conducted at the agro-industrial farm«Niva» (Novonikolaevka village, Dnipropetrovsk region), in which the enzyme preparation «Celloviridin Dx20», which promotes the breakdown of plant cell polysaccharides (cellulose, xylans) was introduced to a standard feed mixture used in swine husbandry. The composition of the ration consisted of 60% barley, 20% wheat and 20% of the protein-vitamin-mineral complex «ShenPigStart». The results of the experiment showed that the introduction into the feed mixture of the enzyme preparation at 100 g per ton helps to increase the body weight of pigs by 11.6% compared to the control, can increase the digestion rate of fibre (13.8%), protein and fat. These results can be explained by the mechanism of action of the enzyme complex, which consists in the hydrolysis of polysaccharides of plant cell walls in the feed in the digestive tract of pigs, the formation of digestable polysaccharides and the release of additional quantities of intracellular proteins. The result of these phenomena is also a positive trend in feed nitrogen assimilation in animals of the experimental group: a 19% decrease in the amount of physiological secretions and a 12.5% increase in nitrogen use. At the same time hematology (hemoglobin, erythrocytes, leukocytes), calcium and phosphorus metabolism remained unchanged. This shows that the preparation used does not accumulate in the body and gradually loses enzymatic activity, and under the influence of the pigs’ stomach proteases it is broken down into separate amino acids which are absorbed by the animals. The resulting stimulatory effect of using "Celloviridin Dх20" has not only physiological but also economic importance, as it helps reduce the time for the pigs to reach slaughter weight (close to 100 kg) by 11 days, and consequently, reduces feed intake and its costs in rearing pigs on farms. Therefore, it is advisable to use "Celloviridin Dх20" in swine husbandry.

Effect of dietary genistein on growth performance, digestive enzyme activity, and body composition of Nile tilapia Oreochromis niloticus

An 8-week feeding experiment was performed to evaluate the effect of dietary genistein on growth performance, body composition, and digestive enzymes activity of juvenile Nile tilapia (Oreochromis niloticus). Four isonitrogenous and isoenergetic diets were formulated containing four graded supplements of genistein: 0, 30, 300, and 3 000 μg/g. Each diet was randomly assigned in triplicate to tanks stocked with 15 juvenile tilapia (10.47±1.24 g). The results show that 30 and 300 μg/g dietary genistein had no significant effect on growth performance of Nile tilapia, but the higher level of genistein (3 000 μg/g) significantly depressed the final body weight and specific growth rate. There was no significant difference in survival rate, feed intake, feed efficiency ratio or whole body composition among all dietary treatments. An assay of digestive enzymes showed that the diet containing 3 000 μg/ggenistein decreased stomach and hepatopancreas protease activity, and amylase activity in the liver and intestine, while a dietary level of 300 μg/g genistein depressed stomach protease and intestine amylase activities. However, no significant difference in stomach amylase activity was found among dietary treatments. Overall, the results of the present study indicate that a high level of dietary genistein (3 000 μg/g, or above) would significantly reduce the growth of Nile tilapia, partly because of its inhibitory effect on the activity of major digestive enzymes. Accordingly, the detrimental effects of genistein, as found in soybean products, should not be ignored when applied as an alternative ingredient source in aquaculture.

Alterations in Digestive Enzymes of Three Freshwater Teleostean Fishes by Almix Herbicide: A Comparative Study

Three freshwater teleostean fishes viz., Anabas testudineus (Bloch), Heteropneustes fossilis (Bloch) and Oreochromis niloticus (Linnaeus) were exposed to almix (66.67 mg/l) herbicide for 30 days to investigate the activity of digestive enzymes (amylase, lipase and protease) in stomach, intestine and liver. Amylase activity showed significantly high (p A. testudineus > H. fossilis.

Dramatically Increased pH and Temperature Stability of Chymotrypsin Using Dual Block Polymer-Based Protein Engineering

In this study, we report on multimodal temperature-responsive chymotrypsin-poly(sulfobetaine methacrylamide)-block-poly(N-isopropylacrylamide) (CT-pSBAm-block-pNIPAm) protein-polymer conjugates. Using polymer-based protein engineering (PBPE) with aqueous atom transfer radical polymerization (ATRP), we synthesized three different molecular weight CT-pSBAm-block-pNIPAm bioconjugates that responded structurally to both low and high temperature. In the block copolymer grown from the surface of the enzyme, upper critical solution temperature (UCST) phase transition was dependent on the chain length of the polymers in the conjugates, whereas lower critical solution temperature (LCST) phase transition was independent of molecular weight. Each CT-pSBAm-block-pNIPAm conjugate showed temperature dependent changes in substrate affinity and productivity when assayed from 0 to 40 °C. In addition, these conjugates showed higher stability to harsh conditions, including temperature, low pH, and protease degradation. Indeed, the PBPE-modified enzyme was active for over 8 h in the presence of a stomach protease at pH 1.0. Using PBPE, we created a dual zone shell surrounding each molecule of enzyme. The thickness of each zone of the shell was engineered to be separately responsive to temperature.

Effects of supplemental Azomite in diets on growth, immune function and disease resistance of white shrimp (Litopenaeus vannamei)

This study was conducted to evaluate effects of dietary Azomite, a natural mineral of volcanic ash, on growth performance, digestive enzyme activities, serum non-specific immunity and disease resistance of white shrimp (Litopenaeus vannamei). Graded levels of 0.0 g kg−1 (control group), 2.0, 4.0, 6.0 and 8.0 g kg−1 Azomite were supplemented in basal diet to feed shrimps. After 6 weeks feeding, weight gains of the 2.0 and 4.0 g kg−1 Azomite groups were significantly higher than that of the control group (P < 0.05). Compared with control group, the activities of stomach protease, hepatopancreas lipase, and serum alkaline phosphatase, lysozyme and phenoloxidase were significantly increased by the 4.0 g kg−1 Azomite addition (P < 0.05). When subjected to hypoxia stress, the lethal times of the first shrimp and all shrimps were both delayed by the addition of 2.0–6.0 g kg−1 Azomite to diets, compared with the lethal time of control group. At the 96th hour after the injection of bacterium (Vibrio alginolyticus), the accumulated mortality of 4.0 g kg−1 Azomite addition group was 34.6% lower than that of control group (P < 0.05). The results showed that dietary levels of 2.0 and 4.0 g kg−1 Azomite can improve the growth performance, digestive enzyme activities, serum non-specific immune function and disease resistance of white shrimp.

Digestive enzymes activity of wild and cultivated grey mullet Mugil cephalus

Digestive tract indices and digestive enzyme distribution in digestive organs of wild and cultivated grey mullet Mugil cephalus were studied,and effects of different temperature and pH on digestive enzyme activities were investigated in both populations.The results showed that in wild and cultivated grey mullets,the ratio of intestine gut length to body length were 2.886±0.301 and 3.315±0.141 while the ratios of viscera,liver and stom-ach weight to body weight were 0.043±0.001 and 0.048±0.004,0.009±0.001 and 0.007±0.000,0.008±0.002 and 0.007±0.000,respectively.The activities of protease in different organs were sequenced as follow: hind-gutforegutmidgutstomachhepatopancreas in wild grey mullets and foregutmidguthindgut hepato-pan-creasstomach in cultured ones,while the activities of amylase were in the order of hindgut pyloric caecummidgutforeguthepatopancreasstomach in wild grey mullets and pyloric caecum foregutmidgut hindgutstomach hepatopancreas in cultured ones.The differences of amylase activity in every part between the populations were extremely significant(P0.01).No significant differences of protease activity in hepatopancreas and stomach exited between the two populations(P0.05),but in midgut the difference was significant(P0.05),and in foregut and hindgut,the difference were extremely significant(P0.01).For the amylase in wild grey mullet,the optimum temperatures were 40℃,45℃,45℃,45℃,40℃,45℃ in hepatopancreas,stomach,foregut,midgut,hindgut and pyloric caecum,respectively and the optimum pH in hepatopancreas was 6.2 and in other tissues was 7.2.For amylase in cultured grey mullet,the optimum temperatures were 40℃,40℃,40℃,45℃,40℃,40℃ in hepatopancreas,stomach,foregut,midgut,hindgut and pyloric caecum,respectively and the optimum pH in stomatch and foregut were 2.2 and 8.0,and in other tissues was 7.2.For protease in wild grey mullet,the optimum temperature in hepatopancreas,stomach,foregut and hindgut was 45℃,while in midgut was 50℃ and the opti-mum pH of protease in stomach was 3.2,and in other tissues was 7.2.For protease in cultured grey mullets,the optimum temperature in hepatopancreas,stomach and midgut was 40℃,while in foregut and hindgut was 45℃ and the optimum pH of protease in stomach was 3.2,and in other tissues was 7.2.This study shows that in aqua-culture we should consider not only the assimilation of protein,but also that of saccharide.Adjustment of the amount and proportion of amylase and protein according to environmental conditions could promote production and save cost.

Effects of light intensity on husbandry parameters, digestive enzymes and whole-body composition of juvenileEpinephelus coioidesreared in artificial sea water

To provide a specific lighting system to aquaculture, a completely randomized design with five light treatments with three replicates was used to investigate effects of light intensity on juvenile Epinephelus coioides reared in artificial sea water. The weight gain rate (WG, %), specific growth rate (SGR % day−1), protein efficiency ratio (PER) and survival rate (S, %) were significantly higher at the 320–1150-lx treatments (P ≤ 0.05) compared with the other treatments, but the highest food conversion ratio (FCR) was at the 0-lx treatment and the lowest at 600–1150 lx. Total protease, amylase and lipase activities in liver, stomach and intestine were significantly higher at 320–1150-lx conditions and significantly lower at 0 lx (P ≤ 0.05) than in the other treatments. The highest contents of crude protein and crude lipid were observed at 600–1150 lx and 320–550 lx respectively. However, both moisture and ash were highest at 0 lx and lowest at 600–1150 lx. The results indicated that light intensity could significantly affect husbandry parameters, digestive enzymes and whole-body composition of juvenile E. coioides, with optimal light intensity being at 600–1150 lx.

In vitro assessment of the effects of phytate and phytase on nitrogen and phosphorus bioaccessibility within fish digestive tract

The aim of the present work was to assess different possible interactions between phytate (IP6), dietary protein and digestive proteases, as well as the preventive effect of phytase in the formation of protein-IP6 complexes, through the use of a gastrointestinal model simulating conditions existing in the digestive tract of rainbow trout ( Oncorhynchus mykiss). Two commercial phytases, expressed in P. lycii and E. coli, were evaluated. Fungal phytase showed an optimum at pH 5.5 and bacterial phytase showed two peaks at pH 2.5 and 4.5. These phytases evidenced different resistance against gastric proteolytic degradation; E. coli phytase retained 90% of its initial activity after 4 h incubation in the presence of either porcine pepsin or fish stomach proteases, while the dephosphorylation activity of P. lycii phytase was reduced by 90% (P<0.001). The presence of IP6 determined a reduction of 80% in protein solubility (P<0.001) when casein was used as substrate of fish acid proteases. The formation of IP6-protein complexes resulted in lower amino acids liberation from casein (P<0.05). Addition of phytase to soybean meal prevented the formation of such IP6-protein complexes and improved amino acid release by 60% (P<0.01). In contrast, IP6 had not effect on protein solubilisation or amino acid liberation during the intestinal digestion. Under conditions simulating gastric digestion a great amount of IP6 was combined to either porcine pepsin or soluble protein in fish stomach crude extract, precipitating 68 and 100% of such proteins, respectively. A similar, but lower reduction in soluble protein from either pancreatin or fish intestinal extract was observed when simulating intestinal digestion (47 and 33%, respectively). The formation of IP6-protein complexes reduced the activity of all gastric and intestinal proteases assayed (P<0.01) with the exception of chymotrypsin. A positive effect (P<0.05) of low pH on IP6-dephosphorylation was measured when the effect of bacterial phytase (2500 FTU/kg) on the release of soluble P from native IP6 present in soybean meal was tested under different digestion temperatures (6 and 16 °C) and pH (2.0, 3.0 and 4.0). A positive effect of temperature was also observed when the reaction was carried out at pH 2.0 and 3.0 (P<0.001), but not at 4.0, suggesting the existence of a limit in the solubility of IP6 and therefore in its availability for phytase action at this last pH value.

In vivo fluorescence imaging of exogenous enzyme activity in the gastrointestinal tract

Exogenous enzymes are administered orally to treat several diseases, such as pancreatic insufficiency and lactose intolerance. Due to the proteinaceous nature of enzymes, they are subject to inactivation and/or digestion in the gastrointestinal (GI) tract. Here we describe a convenient fluorescence-based assay to monitor the activity of therapeutic enzymes in real time in vivo in the GI tract. To establish the proof of principle, the assay was applied to proline-specific endopeptidases (PEPs), a group of enzymes recently proposed as adjuvant therapy for celiac disease (a highly prevalent immunogenetic enteropathy). A short PEP-specific peptide sequence which is part of larger immunotoxic sequences of gluten was labeled with a fluorescent dye and a corresponding quencher. Upon enzymatic cleavage, the fluorescence emission was dequenched and detected with an in vivo imaging system. PEPs originating from Flavobacterium meningosepticum (FM) and Myxococcus xanthus (MX) were evaluated after oral administration in rats. While MX PEP could not cleave the peptide in the stomach, FM PEP showed significant gastric activity reaching 40-60% of the maximal in vivo signal intensity. However, both enzymes produced comparable fluorescence signals in the small intestine. Coadministration of an antacid drug significantly enhanced MX PEP's gastric activity due to increased pH and/or inhibition of stomach proteases. With this simple procedure, differences in the in vivo performance of PEPs, which could not be identified under in vitro conditions, were detected. This imaging assay could be used to study other oral enzymes in vivo and therefore be instrumental in improving their therapeutic efficiency.