Chymotrypsin Activity Research Articles

Effect of feeding plant-based diet to broodstock on the utilization of plant-based diet in the offspring of rainbow trout Oncorhynchus mykiss

This study investigated the effects of nutritional history during the maturation of rainbow trout Oncorhynchus mykiss on the utilization of a plant-based grower diet in the offspring at the juvenile stage. Fishmeal-based (BF) and plant-based (BP) broodstock diets were fed to an F2 line selectively-bred with plant-based diets (Selected) and to the counterpart line (Control) from two months before the start of gonadal development until the end of ovulation (2 × 2 design). Compared to broodstock fed BF, ovulation tended to be delayed in broodstock fed BP, while nutrient composition of the ovulated eggs and egg quality such as hatchability were similar among treatments. From first feeding, offspring were reared on fishmeal-based diets until they reached approximately 6 g body weight (BW). Then, juveniles of each group (Control-BF, Control-BP, Selected-BF, Selected-BP) were fed a plant-based grower diet (GP) to apparent satiation for 8 weeks at 16 °C (3 tanks/treatment). Specific growth rates (% BW/day) of the Selected groups were significantly higher than the Control groups without a significant effect of broodstock diet. Relative feed intake (% BW/day) of the Selected groups and BP groups were significantly higher than the Control groups and BF groups, respectively. Feed efficiency ratios of the Selected groups and BF groups were significantly higher than the Control groups and BP groups, respectively. Whole body manganese and zinc concentrations of the Selected groups were significantly lower than the Control groups. Activities of lipase, trypsin and chymotrypsin in the intestinal digesta, obtained under restricted feeding (2% BW/day), of juveniles from the Selected groups were significantly higher than those from the Control groups, and the activities of lipase and chymotrypsin of juveniles from the BP groups were also significantly higher than those from the BF groups. These results suggest that the nutritional history of rainbow trout broodstock affects the appetite and digestive physiology of the offspring, but minimally affects their growth. Further fortification of nutrients such as microminerals to plant-based grower diets may be necessary to take full advantage of the effect of selective breeding of rainbow trout with plant-based diets, including the effect of feeding them to the broodstock.

Low particle concentrations of nanoplastics impair the gut health of medaka

The environmental occurrence of nanoplastics (NPs) is now evident but their long-term impacts on organisms are unclear, limiting ecological and health risk assessment. We hypothesized that chronic exposure to low particle concentrations of NPs can result in gut-associated toxicity, and subsequently affect survival of fish. Japanese medaka Oryzias latipes were exposed to polystyrene NPs (diameter 100 nm; 0, 10, 104, and 106 items/L) for 3 months, and histopathology, digestive and antioxidant enzymes, immunity, intestinal permeability, gut microbiota, and mortality were assessed. NP exposures caused intestinal lesions, and increased intestinal permeability of the gut. The trypsin, lipase, and chymotrypsin activities were increased, but the amylase activity was decreased. Oxidative damage was reflected by the decreased superoxide dismutase and alkaline phosphatase and increased malondialdehyde, catalase, and lysozyme. The integrated biomarkers response index values of all NP-exposed medaka were significantly increased compared to the control group. Moreover, NP exposures resulted in a decrease of diversity and changed the intestinal microbiota composition. Our results provide new evidence that long-term NPs exposure impaired the health of fish at extremely low particle concentrations, suggesting the need for long-term toxicological studies resembling environmental particle concentrations when assessing the risk of NPs.

Solid-State Fermentation of Distiller’s Dried Grains with Solubles Improves Digestibility for European Seabass (Dicentrarchus labrax) Juveniles

Aquaculture requires new, economical, and eco-friendly protein sources to replace traditional fisheries and plant ingredients. Using agriculture by-products as protein sources would reduce land-based feed production pressure and waste production, promoting a circular economy and sustainable aquaculture. Distiller’s dried grains with solubles (DDGS) is the main by-product of bioethanol production. Corn DDGS has a high protein level, but its high fiber content limits its use as a feed ingredient, particularly for carnivorous fish. Solid-state fermentation (SSF) uses lignocellulosic-rich substrates, such as DDGS, for microbial growth in the near absence of water, promoting enzyme production that degrades the lignocellulosic matrix, increasing free reducing sugars, protein, and antioxidant levels of the substrate. In the present work, the SSF of corn DDGS with Aspergillus carbonarius, A. ibericus, and A. uvarum was tested. Then, the digestibility of the most promising fermented DDGS (in terms of upgraded nutritional composition) was tested by including it in a reference diet (70% of a reference diet; 48% crude protein; 15% crude lipids) for European seabass (Dicentrarchus labrax) juveniles (171 g averaged weight; trial duration of 52 days). Among the fungi tested, Aspergillus ibericus led a generally higher upgrading of the DDGS nutritional composition, leading to a high amount of protein (from 42.7 to 49.7 g N/kg DM), phenolic compounds (1.49 to 4.86 mg/g caffeic acid equivalents), free sugars (9.5 to 31.9 mg/g), and enzyme production (45 U/g and 68 U/g of cellulase and xylanase, respectively), and a high reduction in acid detergent fiber and neutral detergent fiber content (up to 29 and 43%, respectively). Compared to the unfermented DDGS, fermented DDGS presented increased protein, lipids, starch, and energy digestibility, while phosphorous digestibility was similar. Compared to the reference diet, dietary inclusion of unfermented or fermented DDGS increased trypsin and chymotrypsin activities. The activity of digestive enzymes was not affected by the inclusion of fermented DDGS, except for amylase activity, which was lower with the fermented DDGS than with the unfermented DDGS diet. In conclusion, SSF of DDGS enhanced its nutritional value, increasing DDGS digestibility when included in diets for European seabass juveniles.

Effects of coated folic acid and coated methionine on growth performance, nutrient digestibility and rumen fermentation in Simmental bulls

In one-carbon metabolism, methionine (Met) is methyl donor and folic acid (FA) is responsible for the transfer of methyl groups. Approximately 25 % of coated methionine (CMet) and coated folic acid (CFA) released in the rumen at 24 h of incubation. Therefore, the present study investigated the effects of CMet or/and CFA on growth performance, nutrient digestibility and rumen fermentation in bulls. Forty Simmental bulls, 464 ± 15.9 kg of body weight (BW), were blocked by BW and randomly assigned to four groups in a 2 × 2 factorial arrangement of treatment, with factors being CFA 0 or 6 mg FA/kg dietary dry matter (DM) and CMet 0 or 0.66 g Met/kg dietary DM. The experiment period was 81 days long with 20 days for adaptation and 61 days for data and sample collection. Dry matter intake increased (P = 0.001) with CFA or CMet addition. The CFA × CMet interaction was noticed on average daily gain (ADG; P = 0.001) and feed conversion ratio (FCR; P = 0.020); the improvements in ADG and FCR were greater for supplementing CMet in diets with CFA than in diets without CFA. The digestibility of DM, organic matter, neutral detergent fiber and acid detergent fiber increased (P < 0.050) with CFA or CMet addition. When CMet was supplemented, crude protein digestibility increased in CFA diets and was unchanged in diets without CFA. Ruminal pH decreased (P < 0.050), but total volatile fatty acid concentration increased (P < 0.050) with CFA or CMet addition. Acetate to propionate ratio was unchanged with CFA addition, but decreased (P = 0.039) with CMet addition. Rumen ammonia-N concentration decreased (P < 0.050) for either CFA or CMet addition. The activities of carboxymethyl cellulase, protease, α-amylase and cellobiase and populations of protozoa, total bacteria, fungi, dominant cellulolytic bacteria, Ruminobacter amylophilus and Butyrivibrio fibrisolvens increased (P < 0.050) with CFA or CMet supplementation. The CFA × CMet interaction was noticed on Ruminococcus albus population (P = 0.014); when CMet was supplemented, R. albus population increased for diets without CFA and was unchanged for diets with CFA. The activities of chymotrypsin in duodenum increased (P < 0.050) with CFA or CMet supplementation, and that of amylopsin and trypsin in ileum increased (P < 0.050) for bulls receiving CFA addition. The concentrations of glucose, albumin and urea nitrogen in blood were unchanged and homocysteine decreased (P < 0.050) with CFA or CMet supplementation. The CFA × CMet interaction was noticed on blood total protein concentration (P = 0.045); when CMet was added, blood total protein concentration increased for diets without CFA and was unchanged for diets with CFA. The concentration of folate in blood increased (P = 0.001) for CFA addition. The concentration of Met increased (P = 0.002) for CMet supplementation. The results indicated that dietary CFA or/and CMet supplementation improved growth performance, nutrient digestibility and rumen fermentation, and the greatest daily gain was observed for combined addition of CFA and CMet in bulls.

Effects of dietary nucleotide and yeast cell wall on growth performance, feed utilization, anti-oxidative and immune response of grass carp (Ctenopharyngodon idella)

A 70-day feeding trial was conducted to study the effects of dietary nucleotide, yeast cell wall (containing 20% β -glucan) and their combination on growth performance, feed utilization and immune response of grass carp (Ctenopharyngodon idella) with 69.97 ± 0.05 g of initial body weight. Four isonitrogenous (about 38% crude protein) and isolipidic (about 5% crude lipid) diets were established. Based on the control diet (CD), the other three experimental diets were prepared by adding 0.01% of nucleotide (NT), 0.1% of yeast cell wall (YCW) and NT (0.01%) +YCW (0.1%), respectively. Results showed that no significant difference was found in survival of grass carp ranging from 94.44% to 97.78% among all the groups (P > 0.05). Compared with the control group, weight gain rate, muscle crude protein content, serum protein, trypsin and chymotrypsin activities in midgut, lysozyme and immunoglobulin M in serum significantly increased in fish fed the YCW diet (P < 0.05). The significantly highest weight gain rate, villus height and digestive enzyme activities in midgut and innate immune parameters in serum were found in fish fed the NT + YCW diet (P < 0.05). The gene expressions of β-defensin, hepcidin, il-10 and tgf-β1 in the midgut, and tor and s6k1 in liver significantly increased in fish fed the NT + YCW diet. Meanwhile, the gene expressions of il-1β and tnf-α in the midgut decreased significantly (P < 0.05). The liver histology showed the better development in dietary NT and/or YCW supplemented groups than those in the control group. In conclusion, combination of dietary NT and YCW had significantly synergetic improvements on the growth, feed utilization, digestive enzymes, innate immunity and histology of midgut and liver of grass carp.

Fermented Chinese Herbal Medicine Promoted Growth Performance, Intestinal Health, and Regulated Bacterial Microbiota of Weaned Piglets.

To investigate the effects of fermented Chinese herbal medicine on growth performance, diarrhea rate, nutrient digestibility, and intestinal health of weaned piglets, and to provide the theoretical basis for applying fermented Chinese herbal medicines to weaned piglet production, a total of 162 weaned and castrated piglets at 25 days of age (Duroc × Landrace × Yorkshire, half male and half female) with an initial body weight of 7.77 ± 0.03 kg were randomly divided into the following three groups according to the principle of similar body weight: basal diet (CON) group, basal diet + 3 kg/t fermented Chinese herbal medicine (LFHM) group, and basal diet + 5 g/kg fermented Chinese herbal medicine (HFHM) group. Each group underwent six replicates and there were nine piglets in each replicate. The experiment lasted 24 days, i.e., 3 days for preliminary feeding, and 21 days for the experiment. From Day 1 of the experiment, the piglets were observed and recorded for diarrhea each day. As compared with the CON group, the results indicated: Following the addition of fermented Chinese herbal medicine, the piglets in the LFHM and HFHM groups increased final weight (FW); average daily feed intake (ADFI); average daily gain (ADG) (p < 0.01); apparent digestibility of crude protein (CP) (p < 0.05); as well as chymotrypsin, α-amylase, and lipase activities (p < 0.01). In addition, α-amylase activity in the LFHM group was higher than that in the HFHM group (p < 0.05); chymotrypsin activity in the LFHM group was lower than that in the HFHM group (p < 0.05); as compared with the CON group, the LFHM and the HFHM increased villus height (VH) and crypt depth (CD) in piglet jejunum; isovaleric acid concentration with the HFHM was higher than those with the CON and the LFHM (p < 0.05), but butyrate concentration with the HFFM was lower than those with the CON and the LFHM (p < 0.05). The high-throughput 16S rRNA sequencing of intestinal microbiota results showed that the LFHM and the HFHM affected the microbial α diversity index in weaned piglet colon (p < 0.01). In conclusion, fermented Chinese herbs can improve the growth performance of weaned piglets by promoting the secretion of intestinal digestive enzymes, changing intestinal microbial diversity, regulating the contents of intestinal short chain fatty acids (SCFAs), promoting intestinal health, and improving nutrients digestibility.

A meta-analysis for assessing the contributions of trypsin and chymotrypsin as the two major endoproteases in protein hydrolysis in fish intestine

For the majority of fish species, regardless of being gastric or agastric, trypsin and chymotrypsin are known as the two main alkaline proteases responsible for the initial stage of protein hydrolysis in the fish intestine. Although the critical role of these proteases for protein hydrolysis in fish intestine is without doubt, the relative input of each enzyme in protein hydrolysis is still unclear. Data used in the present study has been retrieved from a bibliographic search using the Dimensions application (https://app.dimensions.ai/discover/publication tool). Retrieved articles were carefully inspected to identify whether they contained the description of the development of ontogenetic activities for trypsin, chymotrypsin, and total alkaline proteases in fish intestine. From the list of consulted articles, 21 studies were chosen based on correlation coefficients (Pearson correlation test), and four groups of fish were identified with high significant correlation between 1) the activity of chymotrypsin and total alkaline proteases; 2) the activity of trypsin, chymotrypsin, and total alkaline proteases; 3) the activity of trypsin and total alkaline proteases, and 4) mainly negative correlation between trypsin, chymotrypsin, and total alkaline proteases. These results indicated that the relative inputs of trypsin and chymotrypsin in protein hydrolysis may vary significantly among different fish species, which is a crucial point for proper understanding of species-specific digestive traits in both natural and aquaculture scenarios.

Evaluation of Liquid Organic Acids on the Performance, Chyme pH, Nutrient Utilization, and Gut Microbiota in Broilers under High Stocking Density.

This study aimed to evaluate the efficacy of organic acids (OAs) in starter broilers and to investigate whether supplemental OAs could alleviate the high stocking density (HSD) stress condition in grower broilers. A total of 408 1-day-old Arbor Acres broilers were assigned into two groups without or with liquid OAs in the starter phase. In the grower phase, each group in the starter phase was divided into a normal stocking density and an HSD. The OA dose was 0.16% at the starter and grower phases. The results showed that at the starter phase, OAs decreased the chyme pH in gizzard and duodenum and increased the activities of chymotrypsin and α-amylase in the duodenal chyme (p &lt; 0.05). In the grower phase, an HSD decreased the growth performance and the ether extract digestibility (p &lt; 0.01). The supplementation of OAs decreased the chyme pH in the gizzard, proventriculus, and duodenum and increased the lipase and α-amylase activities (p &lt; 0.05). The supplemental OAs increased the dry matter and total phosphorous digestibility and the contents of acetic acids, butyric acids, isovaleric acids, and valeric acids (p &lt; 0.05). For cecal microbial compositions at the genus level, an HSD decreased the relative abundance of Blautia, Norank_f__norank_o__RF39, and Alistipes, while supplemental OAs increased the relative abundance of Norank_f__norank_o__RF39 (p &lt; 0.05). In conclusion, although there were no interaction effects between OAs and stocking densities in the present study, it was clear that the supplementation of OAs has beneficial effects on the chyme pH, enzymes activities, and nutrient digestibility in broilers, while an HSD existed adverse effects on the growth performance, nutrient digestibility, and gut microbiota balance in grower broilers.

Effects of Artemisia annua L. Water Extract on Growth Performance and Intestinal Related Indicators in Broilers.

Artemisia annua L. is a natural herb with a variety of bioactive substances, which can play a variety of biological functions such as anti-inflammatory, antioxidant, antibacterial and antiviral, and can be used as a potential feed additive. The purpose of this study was to investigate the effects of different doses of Artemisia annua L. water extract (AAWE) on growth performance and intestinal related indicators in broilers. A total of 200 one-day-old Arbor Acre broilers were selected and randomly divided into five treatment groups, with five replicates in each group and eight birds per replicate. The control group was fed a basal diet, whereas the other groups were fed a basal diet supplemented with 0.5, 1.0, 1.5, or 2.0 g/kg AAWE. On d 21, with the increase in AAWE dose, final body weight and feed efficiency showed a quadratic increase effect, whereas feed intake showed a linear reduction effect; however, the apparent metabolic rate of dry matter, crude protein, and ether extract increased quadratically on d 42. In addition, the activity of duodenal chymotrypsin and trypsin, and of jejunal lipase quadratically increased, whereas the intestine crypt depth linearly decreased on d 42. The number of total anaerobic bacteria increased quadratically, whereas the number of Escherichia coli decreased quadratically. The number of Lactobacillus increased linearly, whereas H2S emission linearly decreased on d 21; moreover, NH3 emission (24 h) quadratically decreased on d 42. In conclusion, AAWE promoted the growth performance and intestinal related indicators of broilers.

Production of mycelial biomass, proteases and protease inhibitors by Ganoderma lucidum under different submerged fermentation conditions.

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.

Uncovering the physiological impacts of soybean meal replacement by Narbonne vetch (Vicia narbonensis) meal in rainbow trout (Oncorhynchus mykiss) diets: Towards the future and sustainable European aquaculture.

The identification and implementation of raw materials to replace fish meal (FM) or its major alternative, soybean meal (SBM), among other factors, is crucial for the sustainable growth of aquaculture. Narbonne vetch (Vicia narbonensis) meal (NVM) has previously been identified as a promising alternative raw material locally produced in Europe to replace SBM. Previous work has showed that 33% replacement of SBM by NVM treated with a commercially available phytase (Rovabio® PHY) does not compromise fish growth performance in a 63-day trial. Here, a deeper characterization of the potential use of NVM to replace SBM was performed to unveil any potential undesired physiological impact in rainbow trout (Oncorhynchus mykiss) juveniles with an initial weight of 38.04 ± 0.07 g. After a 63-day feeding trial, high (66%) SBM replacement by NVM (even when treated with Rovabio® PHY) had a negative impact on rainbow trout: decreasing the activity of alkaline proteases, trypsin, chymotrypsin, and α-amylase; inducing cell shrinkage in hepatocytes; and reducing plasma triglycerides and hepatic vitamin E (VE) levels. No biochemical alterations were observed in blood plasma glucose, cholesterol or redox enzymes, regardless of the % of SBM replacement and/or Rovabio® PHY treatment. In fish fed diets with 33% replacement of SBM by NVM (regardless of being treated with Rovabio® PHY or not), only a reduced content of plasma triglycerides, and a slight modification in the position of the nucleus in enterocytes (more intermediate) from the proximal intestine, were reported. Altered metabolism of both carbohydrates and VE was also confirmed at transcriptional level, possibly being related to the presence of non-starch polysaccharides (NSPs) in NVM. The present study identifies future strategies to improve the use of NVM as an alternative raw material to replace SBM, allowing greater sustainability, and a lower carbon footprint in European aquafeeds to be achieved.

Digestive function and serum biochemical parameters of juvenile Cyprinus carpio in response to substitution of dietary soybean meal with sesame seed (Sesamum indicum) cake

A feeding trial was designed to evaluate effects of soybean meal replacement (SBM) with sesame seed cake (SSC) at 0 (SSC0), 25 (SSC25), 50 (SSC50), 75 (SSC75), and 100 (SSC100) % in isoproteic (30%) and isolipidic (8%) diets on the growth performance, digestive function and serum and hematological parameters in Cyprinus carpio. Four hundred and fifty common carp juveniles (83.09 g) were randomly distributed into 15 tanks with a flow-through system. Triplicate groups of fish were hand-fed one of the diets to visual satiation thrice daily for 90 days. The survival rate, growth performance and somatic indices did not show significant differences between treatments. Among feed utilization indicators, nitrogen retention efficiency was significantly higher in SSC25 than in other treatments. Lipid retention efficiency significantly increased from SSC50 to SSC100. The number of white blood cells was significantly higher in SSC0 than other treatments. Significantly higher hemoglobin was observed in SSC75 as compared to SS0 and SSC25. Serum biochemical parameters did not significantly differ between dietary treatments. Dietary SSC resulted in significant increase in calcium ADC and activity of digestive trypsin, chymotrypsin, lipase, α-amylase, and alkaline phosphatase. Significantly higher villi height and intestinal muscle layer width were observed in fish fed the SSC75 diet. The villi width in SSC25 and SSC50 was significantly less than in SSC0. Comparable growth performance, feed utilization, apparent digestibility, digestive function, hematological and serum factors indicated the possibility of 75–100% substitution of SBM by SSC in the diet of juvenile C. carpio.

De Novo Design of a Highly Selective Nonpeptide Fluorogenic Probe for Chymotrypsin Activity Sensing in a Living System.

Chymotrypsin, an extensively known proteolytic enzyme, plays a substantial role in maintaining physiological functions, including protein digestion, immune response, and tissue repair. To date, intense attention has been focused on the invention of efficient and sensitive chemical tools for chymotrypsin activity measurement. Among them, the "nonpeptide"-based chymotrypsin probe design strategy utilizing the esterase activity of chymotrypsin has been well-developed due to its low cost and high atom-economy feature. However, the ester-bond-based nature of these probes make them possibly vulnerable to esterases and active chemicals. These defects strictly restricted the application of the previously reported probes, especially for imaging in living systems. Therefore, to acquire fluorogenic probes with sufficient stability and specificity for chymotrypsin sensing in a complicated biological environment, a more stable skeleton for nonpeptide-based chymotrypsin probe construction is urgently needed. Herein, a novel nonpeptide-based fluorogenic probe for specific chymotrypsin activity sensing was designed and synthesized by the substitution of an ester-based linker with a heptafluorobutylamide moiety. The acquired probe, named TMBIHF, showed high selectivity toward various enzymes and reactive chemicals, while it retained high sensitivity and catalytic efficiency toward chymotrypsin. Moreover, TMBIHF was successfully applied for monitoring chymotrypsin activity and pancreas development in live zebrafish, specific sensing of exogenous and endogenous chymotrypsin in nude mice, and visualizing chymotrypsin-like activity-dependent cellular apoptosis, thus providing an alternative and reliable way for chymotrypsin-targeted biosensor or prodrug construction.

Ontogenetic Development of Digestive Tract and Enzymes Activity in Hatchery-reared Pink Ear Emperor, Lethrinus lentjan Larvae

Ontogenetic development of the digestive tract and associated organs of Lethrinus lentjan larvae were learned by observing changes in external morphology, histology, and enzymatic changes from hatchling to 40-day post-hatching (dph). Ontogenetic development of the L. lentjan digestive system can be divided into three major stages; the first stage from hatching of larvae to complete absorption of yolk-sac, which ends on 2 dph, the second stage from 3 to 15 dph i.e., from exogenous feeding to the formation of the gastric gland and third stage till completion of metamorphosis (35 dph). Pyloric caeca were developed after 20 dph, which was formed entirely by 27 dph. Enzymatic activities of amylase, lipase, trypsin and chymotrypsin were detected before the onset of exogenous feeding and pepsin was detected from 19 dph onwards in L. lentjan. So considering both histology of digestive tract and enzyme development, weaning of L. lentjan larvae to artificial diet can be practiced after 19-20 dph coinciding with the formation of pyloric caeca and production of pepsin enzyme for better digestibility and consequently better growth and survival. The present study can be expended as a reference guide to understand the digestive tract development and successful hatchery rearing of other economically important marine fishes.

Synthetic peptide array on gold for the electrochemical assessment of the 20S proteasome activity and effect of inhibitory compounds

A sensor for the enzymatic activity and inhibition of the 20S proteasome was developed by immobilizing the synthetic peptide ABZ-VVSYAMG-(O2Oc)2-OH at Au electrodes. The detection principle is based on the electroactivity of ABZ, part of the ABZ-VVSY-OH moiety released from the peptide upon 20S proteasome chymotrypsin action. The peptide was immobilized on a para-amino thiophenol (PATP) self-assembled monolayer on Au electrode by cross-linking its amino group to the -(O2Oc)2-OH moiety of the peptide (Au/PATP/peptide). The immobilization of the peptide and its interaction with 20S proteasome was investigated by SEM, QCM, SPR, ATR-FTIR and electrochemistry. The activity of 20S proteasome was assessed electrochemically by cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) after the immersion Au/PATP/peptide in 20S proteasome solution. CV study showed a decrease in both capacitive and faradaic currents corresponding to the ABZ-VVSY-OH removal, allowing the quantification of the 20S proteasome activity. The EIS study revealed that the resistance corresponding to charge transfer reactions at the peptide/solution interface correlated to the ABZ redox reaction, decreased linearly with increasing the incubation time in 20S proteasome solution. The perfected assay was applied for the investigation of the inhibitory effect of one synthetic, bortezomib, and two naturally occurring, epoxomicin, and lactacystin inhibitors.

Effects of Primary Processing and Pre-Salting of Baltic Herring on Contribution of Digestive Proteases in Marinating Process

The low-technological quality of herring caught during the feeding season makes it impossible to achieve full ripeness of the meat in marinades. One solution may be to assist ripening using herring digestive tract proteases. Therefore, whole herring, headed herring and fillets were marinated for 2–14 days using the German (direct) and Danish (pre-salted) methods. The results showed that the mass of marinades from fillets was lower than from herring with intestines and correlated strongly with salt concentration in the Danish method, in contrast to the German method. Marinades from whole and headed herring had significantly higher trypsin, chymotrypsin, carboxypeptidase-A and cathepsin activities than marinated fillets. The herring marinated with viscera had 2–3 times higher non-protein nitrogen, peptide and amino acid fractions, as well as ripened 3 days faster than the marinated fillets. After 2 weeks of marinating, the fillets did not achieve full ripeness of the meat, unlike marinades made from whole and headed herring. The pre-salting stage in the Danish method significantly reduced cathepsin D activity by the tenth day of marinating, which was compensated by digestive proteases only in the case of whole or headed herring. The digestive proteases activity in the fillets was too low to achieve the same effect. Sensory evaluation of texture and hardness-TPA correlated strongly with several proteases in whole herring marinades, in contrast to a weak correlation with only one protease when marinating fillets. Marinating with intestines makes it possible to produce marinades faster, more efficiently and with higher sensory quality from herring of low-technological quality.

Up-regulated serpin gene involved in Cry1Ac resistance in Helicoverpa armigera

Insect resistance to Bacillus thuringiensis (Bt) is a critical limiting factor for applying the Bt crops. Some studies indicated that decreased protoxin activation because of lower enzymatic activities of trypsin and chymotrypsin and increased expression of serpin might involve in Bt resistance. Our previous study identified an endogenous serpin could inhibit the midgut proteases to activate Cry1Ac and reduce the insecticide activity to Helicoverpa armigera. We hypothesis that up-regulated serpin involve in resistance via inhibiting enzymatic activities of trypsin and chymotrypsin to decrease protoxin activation. Herein, we found the serpin-e gene relative expression in midgut was significantly higher in the LF30 resistant strain than that in the susceptible strain during all developmental stages. Importantly, RNAi-mediated silencing of serpin-e gene expression caused 4.46-fold mortality changes in LF30 strain, but the trypsin and chymotrypsin proteases activities were only changed 0.79-fold and 2.22-fold. In addition, although proteases activities were significantly lower in LF30 strain than that in the susceptible strain, the resistance ratios of LF30 to Cry1Ac protoxin and to activated Cry1Ac toxin were no difference. The results indicated serpins caused insect resistance to Cry1Ac protoxins partly through inhibiting the trypsin and chymotrypsin proteases activities, but it also existed other mechanisms in LF30.

Effects of a Novel Protease on Growth Performance, Nutrient Digestibility and Intestinal Health in Weaned Piglets.

Simple SummaryA novel type of protease with high stability, high resistance to acid and a wider range of enzyme cutting sites has been developed recently. This experiment was conducted to investigate the effects of the novel protease in weaned piglets. Our results indicate that dietary protease supplementation promotes nutrient absorption, improves small intestine morphology and enhances digestive enzyme activities of weaned piglets.This experiment was conducted to evaluate the effects of different protease levels on performance, diarrhea rate, nutrient digestibility, blood metabolites, digestive enzyme activities, and intestinal health of weaned piglets. A total of 96 weaned piglets (Duroc × Landrace × Yorkshire, 28 d of age, initial average BW = 6.55 ± 0.17 kg) were divided into four groups (4 pigs/pen and 6 replicates/group) according to a completely random block design. Piglets were fed different levels of protease (0, 150, 300 or 600 mg/kg of diet) for 28 d. The results showed that the addition of protease had no significant effect on the growth performance of weaned piglets (p > 0.05), and the addition of 300 mg/kg protease significantly increased the apparent total intestinal digestibility (ATTD) of nutrients and the apparent ileal digestibility (AID) of amino acids in weaned piglets (p < 0.05), while the addition of 150 mg/kg and 600 mg/kg protease had no significant effect on the digestibility (p > 0.05). The nutrient digestibility of dry matter (DM), organic matter (OM), crude protein (CP) and total energy (GE) showed a trend of increasing and then decreasing with increasing protease concentration (p < 0.05). Adding 300 and 600 mg/kg protease significantly decreased serum ALB/GLO levels (p < 0.05) and duodenal pH (p < 0.05) and increased duodenal villus height (p < 0.05). The addition of protease significantly increased jejunal trypsin and chymotrypsin activities (p < 0.01) and duodenal and jejunal mucosal tight junction proteins in piglets. The mRNA expression levels of ZO-1 and CLAUDIN-1 in the duodenum together with ZO-1 and OCCLUDIN in the jejunum of piglets in the 300 and 600 mg/kg protease supplementation groups were significantly higher than those in the control group (p < 0.05). The results showed that, compared with the control group, protease could promote nutrient absorption, improve small intestine morphology and enhance digestive enzyme activity in weaned piglets. The suitable addition amount was 150–300 mg/kg in the present study.

Supplemental Enzyme and Probiotics on the Growth Performance and Nutrient Digestibility of Broilers Fed with a Newly Harvested Corn Diet.

Simple SummaryAs an important energy feed material for livestock and poultry, corn is often in short supply and used immediately after harvest. However, newly harvested corn can have anti-nutritional factors (ANFs) and cause animal diarrhea. Feed-grade enzymes and probiotics can help to alleviate ANFs in feedstuffs. Therefore, in this study, several enzymes and a laboratory Pediococcus acidilactici BCC-1, isolated from chick ceca, were added into the newly harvested corn diet for a 21-day trial in broilers. Here, we found that the newly harvested corn diets resulted in shorter digesta emptying time, increased visual fecal water content, and decreased trypsin activity, which may lead to the occurrence of diarrhea in broilers. The supplementation of Pediococcus acidilactici BCC-1 to newly harvested corn diets could improve the activity of trypsin and decrease the FCR (F/G) of broilers. Moreover, supplemental protease could significantly increase the digesta emptying time, reduce the visual fecal water content, and increase trypsin activity, which may relieve the diarrhea of broilers. In conclusion, Pediococcus acidilactici BCC-1 or protease individually, as well as in combination with glucoamylase are recommended in newly harvested corn diets for broilers.A new grain phenomenon happens in newly harvested corn because of its high content of anti-nutritional factors (ANFs), which can cause low nutrient digestibility and diarrhea in animals. Enzymes and probiotics have been shown to relieve the negative effect of ANFs for animals. The purpose of this study was to investigate the effect of enzymes and probiotics on the performance and nutrient digestibility of broilers, fed with newly harvested corn diets. A total of 624 Arbor Acres Plus male broiler chickens were randomly divided into eight treatment groups (A: normal corn diet, CT: newly harvested corn diet, DE: newly harvested corn diet + glucoamylase, PT: newly harvested corn diet + protease, XL: newly harvested corn diet + xylanase, BCC: newly harvested corn diet + Pediococcus acidilactici BCC-1, DE + PT: newly harvested corn diet + glucoamylase + protease, XL + BCC: newly harvested corn diet + xylanase + Pediococcus acidilactici BCC-1). Each group was divided into six replicates, with 13 birds each. On day 21, growth performance, nutrient digestibility, and digestive enzyme activity were measured. Compared with the normal corn diet (PC), the newly harvested corn diet (NC) produced shorter digesta emptying time (p = 0.015) and increased visual fecal water content (p = 0.002) of broilers, however, there was no effect on performance. Compared to the newly harvested corn diet (NC), supplemental enzyme of DE increased the activity of chymotrypsin (p = 0.016), however, no differences in the digestibility of three kinds of organic matter, digesta emptying time, visual fecal water content, or performance were found. Supplemental protease (PT) significantly increased digesta emptying time (p = 0.004) and decreased the activity of maltase (p = 0.007). However, it had no effect on the digestibility of three kinds of organic matter or the performance of broilers. Supplemental xylanase (XL) decreased the activity of amylase (p = 0.006) and maltase (p < 0.001); however, it had no effect on digesta emptying time, visual fecal water content, the digestibility of three kinds of organic matter, or performance of broilers. Supplemental DE, combined with PT (DE + PT), increased the digesta emptying time (p = 0.016) while decreasing the visual fecal water content (p = 0.011), and the activity of amylase (p = 0.011), lipase (p = 0.021), and maltase (p < 0.001), however, there was no effect on performance. Supplemental BCC individually decreased the activity of amylase (p = 0.024) and maltase (p < 0.001), however, it increased the activity of trypsin (p < 0.001) and tended to improve feed conversion ratio (FCR) (p = 0.081). Supplemental BCC-1, combined with XL (XL + BCC), increased the activity of trypsin (p = 0.001) but decreased the activity of amylase (p = 0.013), lipase (p = 0.019), and maltase (p < 0.001). Pediococcus acidilactici BCC-1 (109 cfu/kg), protease (800,000 U/g) individually, or protease (800,000 U/g) in combination with glucoamylase (800,000 U/g) were supplemented in newly harvested corn diets for growing broilers. Hence, this study mainly explores the alleviation effect of enzyme and probiotics on the negative phenomenon caused by the utilization of newly harvested corn in broilers and provides a better solution for the utilization of newly harvested corn in production practice.

The effects of dietary ellagic acid supplementation on growth performance, immune response, antioxidant activity, digestive enzyme activities, and intestinal functions in yellow-feathered broilers.

This study was conducted to investigate the effects of dietary supplementation with ellagic acid (EA) on the performance, immune function, antioxidant activity, digestive enzyme activities, and intestinal functions in yellow-feathered broilers. In total, 288 healthy yellow-feathered broilers with an average body weight of 39 ± 0.24 g were randomly divided into four treatment groups. Broilers were given a corn-soybean meal basal diet supplemented with 0 (control group), 100, 200, or 400 mg/kg EA. In the finisher period and the overall period, the inclusion of 100, 200, and 400 mg/kg EA increased the average daily gain (P < 0.05), and the inclusion of 200 or 400 mg/kg EA decreased the feed/gain ratio compared with the control group (P < 0.05). The best immune activity (immunoglobulin G [IgG] and immunoglobulin M [IgM] concentrations) in serum was shown in the 200 mg/kg EA group (P < 0.05). Broilers fed with 200 or 400 mg/kg EA-containing diets exhibited higher serum catalase and glutathione peroxidase activities (P < 0.05) than control broilers. The inclusion of 200 mg/kg EA in the broiler diets increased intestinal chymotrypsin, pepsin, and lipase activities (P < 0.05). Broilers fed 200 mg/kg EA-containing diets had higher villus height in the jejunum and ileum, a higher ratio between villus height and crypt depth in the jejunum, and a deeper crypt in the duodenum compared to control broilers (P < 0.05). EA reduced the diamine oxidase activity and D-lactate concentration in serum. Furthermore, in birds fed EA-containing diets, the abundance of Rikenella and norank_f_norank_o_Clostridia_UCG-014 in cecum were decreased compared with control birds (P < 0.05). Moreover, in birds fed EA-containing diets, the levels of acetate, butyrate, and total short-chain fatty acids in the cecum were higher (P < 0.05) than those in control birds. These findings indicated that dietary EA had ameliorative effects on antioxidant capability, digestive enzyme activity, immune function, and intestinal functions, which led to strengthened growth performance.