Higher Trypsin Research Articles

Efficiently Substituting Dietary Fish Meal with Terrestrial Compound Protein Enhances Growth, Health, and Protein Synthesis in Largemouth Bass.

Inappropriate substitution of dietary fishmeal (FM) can adversely affect the growth, health, and metabolism of carnivorous fish species. To effectively reduce the amount of dietary FM in carnivorous largemouth bass (Micropterus salmoides), a terrestrial compound protein (Cpro) with chicken meal, bone meal, and black soldier fly protein was used to formulate four isoproteic (52%) and isolipidic (12%) diets, namely T1 (36% FM), T2 (30% FM), T3 (24% FM), and T4 (18% FM), for feeding juveniles (initial weight: ~12 g) for 81 days. Results indicated that the growth performance, feed efficiency, and morphological indicators, as well as muscle texture and edible quality of fish, did not differ significantly among the four groups. However, the muscle protein contents and ATP/AMP ratio of fish in the T4 group were significantly increased in comparison with those of fish in the T1 group, while the opposite was true for muscle glycogen. Compared with the T1 group, high serum total amino acid and MDA contents, as well as low AST activities, were observed in the T3 and T4 groups, and relatively high intestinal trypsin and lipase activities were found in the T2-T4 groups. The transcripts of intestinal proinflammatory cytokines (il-1β, il-6, and tnf-α) were downregulated in the T2-T4 groups compared with T1 group, while the expression of anti-inflammatory cytokines (il-10) and tight junction (zo-1 and occludin) showed the reverse trend. The mRNA expression of positive regulators related to protein synthesis (sirt1, pgc1-α, pi3k, and akt) were significantly upregulated in the muscle of fish fed diets T3 and T4, while their negative regulators (4e-bp1) mRNA levels were downregulated. The results indicate that the dietary FM of largemouth bass could be effectively reduced to at least 18% by the Cpro, which is beneficial to health, digestion, and protein synthesis for maintaining accelerated growth.

Effects of dietary supplementation of Bacillus, β-glucooligosaccharide and their synbiotic on the growth, digestion, immunity, and gut microbiota profile of abalone, Haliotis discus hannai

Probiotics, prebiotics, and synbiotics are known to improve aquatic animals' growth and health conditions. In this study, we investigated the effect of Bacillus sp. KRF-7 (KRF-7), Bacillus sp. PM8313 (PM8313), β-glucooligosaccharide (BGO), and their synbiotics on the growth, digestive physiology, immunity, and gut microbiota of abalone, Haliotis discus hannai. The control diet (CON) was sprayed with 108 CFU/g KRF-7 (PRO1) or PM8313 (PRO2) as probiotics, 0.10% BGO (PRE) as prebiotic, and their combinations (SYN1: PRO1 + BGO; SYN2: PRO2+ BGO) as synbiotic treatments. These diets (triplicates) were given once daily to the abalone (initial body weight: 1.32 ± 0.05 g, initial shell length: 21. 69 ± 0.19 mm) for 12 weeks. The results showed that the PRE diet induced a significant 15% shell length and body weight increase against the CON (SLGR: 45.77% vs. 53.04%; WGR: 182.77% vs. 210.22%), the highest among dietary supplements (P < 0.05). The level of IGF and IGFBP in the cell-free hemolymph, protein expression of IGF signaling, and gene expression of myosin heavy chain in the adductor muscle increased significantly (P < 0.05) following dietary supplementation. Correspondingly, PRE-, PRO1-, and PRO2-fed abalone had the highest trypsin, lipase, and amylase secretion in the hepatopancreas (P < 0.05) and increased histomorphometry (stomach epithelial thickness and intestinal villi height) (P < 0.05). Regarding antioxidative capacity and immunity, the hepatopancreatic expression of SOD, CAT, IRAK-4, NF-Kb, TNF-α, and β-defensin were significantly upregulated in abalone fed with dietary additives compared to the CON (P < 0.05). In terms of gut microbiota profile, PRE-fed abalone had a significant reduction in microbial richness (P < 0.05). The phylum level was similarly occupied, while a diet-specific core microbiota at the genus level was apparent. The findings illustrated that BGO, KRF-7, and PM8313 can be used in abalone aquaculture as growth-promoting and immune-stimulating supplements.

Plectasin: from evolution to truncation, expression, and better druggability.

Non-computational classical evolution analysis of plectasin and its functional relatives can especially contribute tool value during access to meet requirements for their better druggability in clinical use. Staphylococcus aureus is a zoonotic pathogen that can infect the skin, blood, and other tissues of humans and animals. The impact of pathogens on humans is exacerbated by the crisis of drug resistance caused by the misuse of antibiotics. In this study, we analyzed the evolution of anti-Staphylococcus target functional sequences, designed a series of plectasin derivatives by truncation, and recombinantly expressed them in Pichia pastoris X-33, from which the best recombinant Ple-AB was selected for the druggability study. The amount of total protein reached 2.9 g/L following 120 h of high-density expression in a 5-L fermenter. Ple-AB was found to have good bactericidal activity against gram-positive bacteria, with minimum inhibitory concentration (MIC) values ranging between 2 and 16 μg/mL. It showed good stability and maintained its bactericidal activity during high temperatures, strong acid and alkali environments. Notably, Ple-AB exhibited better druggability, including excellent trypsin resistance, and still possessed approximately 50% of its initial activity following exposure to simulated intestinal fluids for 1 h. In vitro safety testing of Ple-AB revealed low hemolytic activity against mouse erythrocytes and cytotoxicity against murine-derived macrophages. This study successfully realized the high expression of a new antimicrobial peptide (AMP), Ple-AB, in P. pastoris and the establishment of its oral administration as an additive form with high trypsin resistance; the study also revealed its antibacterial properties, indicating that truncation design is a valuable tool for improving druggability and that the candidate Ple-AB may be a novel promising antimicrobial agent.

Effects of Dietary Protein Levels on Growth, Digestive Enzyme Activity, Antioxidant Capacity, and Gene Expression Related to Muscle Growth and Protein Synthesis of Juvenile Greasyback Shrimp (Metapenaeus ensis).

An 8-week feeding trial was conducted to assess the effects on growth, antioxidant capacity, digestive enzyme activity, and gene expression related to muscle growth and protein synthesis of juvenile greasyback shrimp (Metapenaeus ensis) using five experimental diets containing 29.37%, 34.30%, 39.11%, 44.05%, and 49.32% of protein. The results demonstrated that juvenile greasyback shrimp consuming 39.11%, 44.05%, and 49.32% dietary protein had a significantly higher final body weight (FBW), weight gain (WG), feed conversion ratio (FCR), and specific growth rate (SGR) than other groups (p < 0.05). The protein efficiency ratio (PER) showed a significantly quadratic pattern with increasing dietary protein levels (p < 0.05). The highest trypsin and pepsin activities were observed in the group with a protein level of 44.05% (p < 0.05). Relatively higher superoxide dismutase (SOD) activity was found in groups with protein levels of 39.11% (p < 0.05). Alkaline phosphatase (AKP) and catalase (CAT) activity showed a significantly linear increasing pattern with increasing protein intake up to 44.05%, and then decreased gradually (p < 0.05). Compared to the dietary 29.37% protein level, the expression levels of myogenic regulatory factors (mef2α, mlc, and myf5) and mTOR pathway (mtor, s6k, akt, and pi3k)-related genes were significantly up-regulated in muscle with 39.11%, 44.05%, and 49.32% dietary protein levels (p < 0.05). The AAR pathway (gcn2, eif2α, and atf4)-related gene expression levels were significantly lower in muscles with 39.11%, 44.05%, and 49.32% protein levels than in other groups (p < 0.05). Based on the broken-line regression analysis of SGR, the estimated appropriate dietary protein requirement for juvenile greasyback shrimp is 38.59%.

Appraising the dose sensitivity of 21d-broiler chicks to a mono-component protease in high trypsin inhibitor soybean-based diet

Exogenous proteases are promoted for their ability to counteract the negative effects of antinutritional elements such as trypsin inhibitors in broiler chickens. This is because these anti-nutritional substances are proteinaceous, making them candidates for hydrolysis by proteases. However, there still is insufficient knowledge on the sensitivity of this phenomena to protease dosing, hence this study was designed to evaluate the dose sensitivity of broiler starter chickens fed a high trypsin inhibitor soyabean-based diet supplemented with a mono-component protease. Two hundred and sixty-four one-day-old chicks were divided into three treatments, each consisting of eight replicates and 11 chicks per replicate. The control group (C) were offered a corn-soybean-based diet contained 8.47 mg/g of trypsin inhibitor, while the other two groups were offered the control diet supplemented with 250 g/tonne (C+250) and 500 g/tonne (C+500) of a mono-component protease, Cibenza DP100, for 21 days. Blood was sampled for serum albumin, uric acid, creatinine, and total protein , and pancreas, liver, and mid-sections of the jejunum, and ileum were harvested for examination of organ and gut morphology. The findings demonstrated that feed conversion ratio improved with protease dosage, with the C+500 group demonstrating the highest efficiency (1.48 ± 0.22). Serum albumin, total protein, and uric acid levels also increased significantly (p&lt;0.05) with birds fed diet C+500 having the highest values. In contrast, serum creatinine was significantly (p&lt;0.05) higher in the control group. Protease supplementation also increased (p&lt;0.05) villi height and crypt depth in both ileum and jejunum. The relative weight of the liver increased (p&lt;0.05) while that of pancreas decreased (p&lt;0.05) with supplemental protease. In conclusion, these findings suggest that supplementation of a high trypsin inhibitor soybean-based diet with a mono-component protease at 500g/tonne complete diet could be used to achieve better performance in 21-d old chickens.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; Les protéases exogènes sont promues pour leur capacité à contrer les effets négatifs des éléments antinutritionnels tels que les inhibiteurs de la trypsine chez les poulets à griller. En effet, ces substances anti-nutritionnelles sont protéiques, ce qui en fait des candidats à l'hydrolyse par les protéases. Cependant, les connaissances sur la sensibilité de ce phénomène au dosage de la protéase sont encore insuffisantes. Cette étude a donc été conçue pour évaluer la sensibilité à la dose de poulets de chair nourris avec un régime à base de soja à haute teneur en inhibiteur de trypsine, complété par une protéase à un seul composant. Deux cent soixante-quatre poussins d'un jour ont été divisés en trois traitements, chacun consistant en huit répétitions et 11 poussins par répétition. Le groupe témoin (C) s'est vu proposer un régime à base de maïs-soja contenant 8,47 mg/g d'inhibiteur de trypsine, tandis que les deux autres groupes se sont vu proposer le régime témoin supplémenté en 250 g/tonne (C+250) et 500 g/tonne (C+500) d'une protéase mono-composant, Cibenza DP100, pendant 21 jours. Le sang a été prélevé pour l'albumine sérique, l'acide urique, la créatinine et les protéines totales, et le pancréas, le foie et les parties médianes du jéjunum et de l'iléon ont été prélevés pour l'examen de la morphologie des organes et de l'intestin. Les résultats ont démontré que le taux de conversion alimentaire s'améliorait avec le dosage de la protéase, le groupe C+500 démontrant l'efficacité la plus élevée (1,48 ± 0,22). Les taux d'albumine sérique, de protéines totales et d'acide urique ont également augmenté de manière significative (p&lt;0,05), les oiseaux nourris au régime C+500 ayant les valeurs les plus élevées. En revanche, la créatinine sérique était significativement (p&lt;0,05) plus élevée dans le groupe témoin. La supplémentation en protéase a également augmenté (p&lt;0,05) la hauteur des villosités et la profondeur des cryptes dans l'iléon et le jéjunum. Le poids relatif du foie a augmenté (p&lt;0,05) tandis que celui du pancréas a diminué (p&lt;0,05) avec la protéase supplémentaire. En conclusion, ces résultats suggèrent que la supplémentation d'un régime à base de soja à haute teneur en inhibiteur de trypsine avec une protéase monocomposant à 500 g/tonne de régime complet pourrait être utilisée pour obtenir de meilleures performances chez les poulets âgés de 21 jours

A comparison of digestive strategies for fishes with different feeding habits: Digestive enzyme activities, intestinal morphology, and gut microbiota.

Fish feeding habit determines the digestive tract structure and intestinal microflora. However, the relationship between feeding habit, digestive intestinal morphology, and microbial diversity of omnivorous, herbivorous, plankton feeder, and carnivorous fish from the same environment has not been compared. This study compared the digestive enzyme activities, intestinal morphology, and intestinal microflora of omnivorous (Carassius auratus), herbivorous (Ctenopharyngodon idellus), carnivorous (Siniperca chuatsi), and plankton feeder (Schizothorax grahami) fishes and predicted the potential functions of specific microflora on different nutrients. Twelve intestine samples were collected from each of the four fishes from Dianchi Lake. The composition and diversity of microbial communities were determined by using high-throughput sequencing of 16S rDNA. The results showed that the carnivorous fish (S. chuatsi) had higher trypsin and pancrelipase activities in the hepatopancreas and enteropeptidase in the intestine, but lower amylase activities in the intestine. The carnivorous fish intestine had more microvilli branches and complex structures than other fish species in the order carnivorous > herbivorous > plankton feeder > omnivorous. The intestinal microflora diversity was higher in the omnivorous fish and followed the order omnivorous > herbivorous > plankton feeder > carnivorous. Acinetobacter species and Bacteroides species were the most dominant flora in the carnivorous and herbivorous fishes, respectively. Acinetobacter species and Pseudomonas species might help the host to digest protein, while Bacteroidetes species may help the host to digest cellulose. Taken together, feeding habit determines the digestive enzyme activities, intestinal tissue morphology, and differential colonization of fish intestinal flora. The knowledge obtained is useful in feed formulation and feeding practices for the studied fish species.

Design of a bifunctional pan-sarbecovirus entry inhibitor targeting the cell receptor and viral fusion protein.

Development of highly effective antivirals that are robust to viral evolution is a practical strategy for combating the continuously evolved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Inspired by viral multistep entry process, we here focus on developing a bispecific SARS-CoV-2 entry inhibitor, which acts on the cell receptor angiotensin converting enzyme 2 (ACE2) and viral S2 fusion protein. First, we identified a panel of diverse spike (S) receptor-binding domains (RBDs) and found that the RBD derived from Guangdong pangolin coronavirus (PCoV-GD) possessed the most potent antiviral potency. Next, we created a bispecific inhibitor termed RBD-IPB01 by genetically linking a peptide fusion inhibitor IPB01 to the C-terminal of PCoV-GD RBD, which exhibited greatly increased antiviral potency via cell membrane ACE2 anchoring. Promisingly, RBD-IPB01 had a uniformly bifunctional inhibition on divergent pseudo- and authentic SARS-CoV-2 variants, including multiple Omicron subvariants. RBD-IPB01 also showed consistently cross-inhibition of other sarbecoviruses, including SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus (PCoV-GX). RBD-IPB01 displayed low cytotoxicity, high trypsin resistance, and favorable metabolic stability. Combined, our studies have provided a tantalizing insight into the design of broad-spectrum and potent antiviral agent. IMPORTANCE Ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution and spillover potential of a wide variety of sarbecovirus lineages indicate the importance of developing highly effective antivirals with broad capability. By directing host angiotensin converting enzyme 2 receptor and viral S2 fusion protein, we have created a dual-targeted virus entry inhibitor with high antiviral potency and breadth. The inhibitor receptor-binding domain (RBD)-IPB01 with the Guangdong pangolin coronavirus (PCoV-GD) spike RBD and a fusion inhibitor IPB01 displays bifunctional cross-inhibitions on pseudo- and authentic SARS-CoV-2 variants including Omicron, as well as on the sarbecoviruses SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus. RBD-IPB01 also efficiently inhibits diverse SARS-CoV-2 infection of human Calu-3 cells and blocks viral S-mediated cell-cell fusion with a dual function. Thus, the creation of such a bifunctional inhibitor with pan-sarbecovirus neutralizing capability has not only provided a potential weapon to combat future SARS-CoV-2 variants or yet-to-emerge zoonotic sarbecovirus, but also verified a viable strategy for the designing of antivirals against infection of other enveloped viruses.

The First Anti-Snakebite and Hepatoprotective Characterization of a Trypsin Kunitz-like Inhibitor (EcTI) from the Plant Enterolobium contortisiliquum; A Case of Two Soul Mates Meeting.

Snake venom serine protease (SVSP) interferes with the regulation and control of important biological reactions in homeostasis and can be classified as an activator of the fibrinolytic system and platelet aggregation. Our group has recently isolated a new serine protease from Crotalus durissus terrificus total venom (Cdtsp-2). This protein exhibits edematogenic capacity and myotoxic activity. A Kunitz-like EcTI inhibitor protein with a molecular mass of 20 kDa was isolated from Enterolobium contortisiliquum and showed high trypsin inhibition. Thus, the objective of this work is to verify the possible inhibition of the pharmacological activities of Cdtsp-2 by the Kutinz-type inhibitor EcTI. To isolate Cdtsp-2 from total C. d. terrificus venom, we used three-step chromatographic HPLC. Using the mice paw edema model, we observed an edematogenic effect, myotoxicity and hepatotoxicity caused by Cdtsp-2. In vitro and in vivo experiments showed that the alterations in hemostasis caused by Cdtsp-2 are crucial for the development of marked hepatotoxicity and that EcTI significantly inhibits the enzymatic and pharmacological activities of Cdtsp-2. Kunitz-like inhibitor may be a viable alternative for the development of ancillary treatments against the biological activities of venoms.

In vitro trypsin inhibitory activities of some plant and fruit extracts and chemical compounds

Trypsin, an enzyme from the serine protease class, is known to be involved in the degradation of proteins. Excessive activity of trypsin is strongly implicated in triggering many diseases, such as acute pancreatitis, inflammation, and tumour. Therefore, this enzyme's regular and balanced activity is necessary for normal physiological functions. Thus, there is a need to develop new trypsin inhibitors from natural sources and chemical compounds. In this study, the inhibitory effects of aqueous extracts prepared from 29 different plants and 10 different chemical compounds were investigated on the activity of trypsin due to its importance in the health sector. The present study's plant extracts and chemical compounds showed trypsin-inhibitory effects. The inhibitory activities of the extracts and chemical compounds increased in a dose-dependent manner. Several plant extracts and chemical compounds that showed high trypsin inhibitory activities may be appropriate for use as trypsin inhibitors to provide additional support to drug treatment in the health field.

ACE2-Independent Bat Sarbecovirus Entry and Replication in Human and Bat Cells.

Hundreds of sarbecoviruses have been found in bats, but only a fraction of them have the ability to infect cells using angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV and -2. To date, only ACE2-dependent sarbecoviruses have been isolated from field samples or grown in the laboratory. ACE2-independent sarbecoviruses, comprising the majority of the subgenus, have not been propagated in any type of cell culture, as the factors and conditions needed for their replication are completely unknown. Given the significant zoonotic threat posed by sarbecoviruses, cell culture models and in vitro tools are urgently needed to study the rest of this subgenus. We previously showed that the exogenous protease trypsin could facilitate cell entry of viral-like particles pseudotyped with spike protein from some of the ACE2-independent sarbecoviruses. Here, we tested if these conditions were sufficient to support bona fide viral replication using recombinant bat sarbecoviruses. In the presence of trypsin, some of the spike proteins from clade 2 viruses were capable of supporting bat sarbecovirus infection and replication in human and bat cells. Protease experiments showed a specific viral dependence on high levels of trypsin, as TMPRSS2 and furin had no effect on clade 2 virus entry. These results shed light on how sarbecoviruses transmit and coexist in their natural hosts, provide key insights for future efforts to isolate and grow these viruses from field samples, and further underscore the need for broadly protective, universal coronavirus vaccines. IMPORTANCE Our studies demonstrate that some unexplored sarbecoviruses are capable of replicating in human and bat cells in an ACE2-independent way but need a high trypsin environment. We found that trypsin is not compensated by other known proteases involved in some coronavirus entry. This work provides important information that the trypsin-dependent entry may be a widely employed mechanism for coronaviruses and will help for further understanding the biological features of the less-studied viruses.

Modeling of Proteolysis of β-Lactoglobulin and β-Casein by Trypsin with Consideration of Secondary Masking of Intermediate Polypeptides.

The opening of protein substrates during degradation by proteases and the corresponding exposure of their internal peptide bonds for a successful enzymatic attack, the so-called demasking effect, was studied for β-lactoglobulin (β-LG) and β-casein (β-CN) hydrolyzed by trypsin. Demasking was estimated by monitoring the redshift in intrinsic tryptophan fluorescence, characterizing the accessibility of polypeptide chains to aqueous medium. The secondary masking of intermediate polypeptides, giving an inverse effect to demasking, caused a restriction of the substrate opening. This led to the limitations in the red shift of fluorescence and the degree of hydrolysis with a long time of hydrolysis of β-LG and β-CN at a constant substrate concentration and reduced trypsin concentrations. The proposed proteolysis model included demasking of initially masked bonds in the protein globule or micelle, secondary masking of intermediate polypeptides, and their subsequent slow demasking. The hydrolysis of peptide bonds was modeled taking into account different hydrolysis rate constants for different peptide bonds. It was demonstrated that demasking competes with secondary masking, which is less noticeable at high trypsin concentrations. Modeling of proteolysis taking into account two demasking processes and secondary masking made it possible to simulate kinetic curves consistent with the experimental data.

Persistence of Digestive Enzyme Activities of Sea Cucumber (Apostichopus japonicus): Evidence From Diet Switch After Evisceration

A two-stage diet-switch experiment was conducted to examine the hypothesis that the changes in digestive enzyme activities of sea cucumber (Apostichopus japonicus) induced by historic diets might persist in the regenerated intestines. In stage I, A. japonicus were treated with two different diets for 56 days, including diet A with 11% crude protein, 1% crude lipid, and 40% carbohydrate, and diet B with 18% crude protein, 2% crude lipid, and 35% carbohydrate. In stage II, each treatment was subjected to evisceration with 0.35M KCl or not (eviscerated and non-eviscerated groups), half of which were then switched to different diets from diet A to B or vice versa for 112 days. The persistence of digestive enzyme activities was evaluated by measuring the changes in digestive enzyme activities before and after evisceration. In stage I, diets B and A increased trypsin and amylase activities, respectively. In stage II, the higher trypsin activities were observed in eviscerated and non-eviscerated A. japonicus that had consumed diet B in stage I. The higher amylase activities were observed only in eviscerated A. japonicus that had consumed diet A in stage I. It indicated that the historic diets showed long-term effects on the digestion of A. japonicus, which led to the persistence of changes in both trypsin and amylase activities in intestines, especially in the regenerated intestines. In addition, the specific growth rates (SGRs) and metabolic rates (MRs) of A. japonicus were affected by the long-term effects of historic diets. Meanwhile, the relationships between enzymic activities, SGRs, and MRs were observed in A. japonicus, indicating that the historic diets could produce long-term effects on the growth and metabolism of A. japonicus through their long-term effects of historic diets on digestive enzyme activities. In conclusion, the present study showed that the changes in digestive enzyme activities induced by different diets in stage I could persist in the intestines and regenerated intestines, leading to long-term effects of historic diets on the growth and metabolism of A. japonicus.

High effective proteinaceous α-amylase inhibitors from grains and control release

Plant-derived α-amylase inhibitors exhibit great capacity in glycaemic control by restraining dietary starch digestion. However, natural α-amylase inhibitors with strong inhibitory activity and good stability are still scarce. In this study, the inhibitory activity and stability of proteinaceous α-amylase inhibitors derived from eleven grains were examined, and microencapsulation was carried out to protect the inhibitors in gastric fluid. We found that the highland barley protein extract (HBPE) showed the most potent α-amylase inhibitory activity (IC50 = 1.11 mg/mL). HBPE is a reversible noncompetitive α-amylase inhibitor and displayed good stability across various pH and temperatures. It showed high trypsin tolerance but low pepsin tolerance. Thus HBPE-encapsulated microspheres were prepared by the composite of sodium alginate/gellan gum with the internal ionic gelation method. The optimum encapsulation condition was determined, and the prepared microspheres displayed discrete, uniform and regular sphericities with diameters around 53 μm, and better mechanical strength with more than 81% activity retention rate. Moreover, the microspheres protected the HBPE in gastric fluid and extended HBPE release in the small intestinal fluid, revealing excellent control release of HBPE in simulated gastrointestinal digestion. Our study provides a new and effective strategy for the management of postprandial blood glucose.

Influence of Hot-water Treatment on the Trypsin Inhibitor Activity Reduction of Sweet Potatoes

HighlightsSweet potatoes designated for animals contains higher trypsin inhibitor activity.The trypsin inhibitor activity of sweet potatoes was reduced by hot-water treatment.The optimal conditions of hot-water treatment were determined to be 95°C and 14 min.Sweet potato extracts are reversible competitive trypsin inhibitors.ABSTRACT.Sweet potatoes are a good food source, but they contain trypsin inhibitors, which reduce the protein utilization rate in animals. This study analyzed the trypsin inhibitor activity of ten sweet potato varieties. Most of the sweet potatoes designated for animals exhibited high trypsin inhibitor activity. The trypsin activities of TNG57 and TNG10, the most popular sweet potato varieties in Taiwan, were 51.5% and 77.1%, respectively. However, the trypsin inhibitor activity of these fresh sweet potatoes was successfully reduced by hot-water treatment. The influence of hot-water temperature and treatment time was analyzed by response surface methodology (RSM). From the RSM regression models, the optimal conditions of hot-water treatment were determined to be 95°C and 14 min. The kinetic parameters (Km and Vmax) of sweet potato extracts were determined from a Lineweaver-Burke plot. The inhibitor in sweet potato extracts was classified as reversible competitive. Keywords: Hot-water treatment, Sweet potato, Trypsin inhibitor, Response surface methodology.

Compensatory growth, antioxidant capacity and digestive enzyme activities of Sobaity ( Sparidentex hasta ) and yellowfin seabreams ( Acanthopagrus latus ) subjected to ration restriction

The compensatory growth response (CGR) and some selected physiological parameters were evaluated in sobaity (Sparidentex hasta, 10 g) and yellowfin seabreams (Acanthopagrus latus, 4.3 g) juveniles subjected to a 2-week restricted feeding. Fish were first fed at 0%, 25%, 50% and 75% of the satiation level for a 2-week period, and then, they were refed for 6 weeks at the visual satiation level. The control group was fed to the satiation everyday for 8 weeks. Three hundred sobaty seabream juveniles were stocked into 15 tanks (20 fish tank−1), and 450 yellowfin seabream juveniles were allocated into 15 tanks (30 fish tank−1) and were fed on a commercial feed (500 g kg−1 crude protein and 150 g kg−1 crude fat). Survival rate was decreased in R0% group in S. hasta because of the cannibalism, which was triggered by feed restriction. After the 2 weeks of feed restriction, the control and R0% treatments in S. hasta (16.0 vs. 9.3 g) and A. latus (3.9 vs. 6.2 g) had the highest and lowest body weight, respectively. After the 6 weeks of refeeding trial, all the ration-restricted groups showed a full CGR in S. hasta. Regarding A. latus juveniles, except for R0%, the other groups showed a full CGR. Feed conversion ratio was improved in S. hasta that subjected to the 2-week feed restriction, but this parameter did not change in A. latus. Antioxidant enzyme activities in the liver of S. hasta juveniles, including glutathione-S-transferase, glutathione peroxidase, superoxide dismutase and catalase along with liver enzymes including alkaline aminotransferase, aspartate amino transferase, lactate dehydrogenase and alkaline phosphatase gradually were decreased with reducing the severity of the feed restriction. Regarding A. latus, all mentioned antioxidant and the liver enzymes in the feed-restricted groups were higher than the control. R75% and control groups in S. hasta had the greatest and the least trypsin and alkaline phosphatase activities. In A. latus, R25%, R50% and R75% groups had higher trypsin, chymotrypsin and lipase activities compared with the other groups (p < .05). By considering all the selected physiological responses in both species, it can be concluded that increasing the feed restriction severity can trigger oxidative stress that may compromise health condition.

Nutrient Digestibility and Digestive Enzyme Activity in Fringe Lipped Carp, Labeo fimbriatus (Bloch, 1795), Fed Diets Containing Cottonseed Meal

Advanced fingerlings of Labeo fimbriatus (Bloch, 1795) were reared in aerated indoor plastic tanks to elucidate the dry matter and nutrient digestibility of cottonseed meal (CSM) in the feed. The fish were fed isocaloric formulated diets containing CSM replacing the main ingredients – groundnut cake and rice bran of the control diet at 10, 20, 30 and 40 % levels. Total dry matter digestibility and major nutrient digestibility were estimated using acid insoluble ash as the marker. In general, the digestibility of dry matter, protein, fat, and nitrogen-free extract was higher in CSM incorporated diets than the control diet. While the dry matter and nitrogen-free extract digestibility values were highest at 40 % CSM incorporation level, those for protein and fat digestibility were highest in 30 % and 10 % levels, respectively, showing an apparent reduction thereafter. The incorporation of CSM had a stimulatory effect on most of the intestinal digestive enzymes tested. Fish fed 20 % CSM diet recorded the highest activity of total protease and carboxypeptidases while those fed 30 % CSM diet had highest trypsin and amylase activities; all showing reducing trend consequently. In the case of hepatopancreas, the reducing trend in the activities of protease, chymotrypsin, carboxypeptidase A, and lipase observed with increased incorporation levels of CSM was significant (P &lt; 0.05) and the reduction in the activities of trypsin, amylase and cellulase in CSM fed fish was not significant (P &gt; 0.05). The study indicates the possibility of incorporation of CSM in the diet of L. fimbriatus up to 40 % level without affecting nutrient digestibility.

Effects of different physical technology on compositions and characteristics of bean dregs

The effects of ultrafine grinding (U), high pressure (HP), microwaves (M), high-temperature cooking (HTC) and combination technologies (U-HP, U-M, U-HTC) on the nutritional compositions and characteristics of bean dregs were investigated. The results showed that both single treatments and combination treatments significantly increased the soluble dietary fiber (SDF) content and water solubility of bean dregs; however, the protein content, fat absorption capacity and swelling capacity of bean dregs were decreased compared with those of the control. The combination technologies significantly increased the contents of K, Ca, Na and Fe in bean dregs. HTC and U-HTC had prominent effects on inhibiting trypsin inhibitor activity, which were decreased from 7365 TIU/g to 1210 and 96 TIU/g, respectively. The bean dregs by ultrafine grinding and combined treatments showed honeycomb structure and small particle distribution, and their processing performances improved. In conclusion, combination technologies were effective methods for improving the quality of bean dregs and expanding their development.Industrial relevance: A combined method may have greater effects than any single approach in improving the quality of bean dregs. Ultrafine grinding technology combined with other physical techniques used in bean dregs can solve quality problems; for example, bean dregs are characterized by poor taste, perishability, low soluble fiber content, and high trypsin inhibitor activity, all of which are related to human health and safety. However, to the best of our knowledge, ultrafine grinding combined with other physical techniques has not been used with bean dregs. The present paper highlights the effects of ultrafine grinding technology (U) combined with high pressure (HP), microwave (M), and high temperature cooking (HTC) technologies on the nutritional and functional compositions of bean dregs. The obtained results contribute to enhance SDF content, reduce anti-nutrition factors, and expand development and utilization of bean dregs, which constitute the basis for its application in baked food, flour products and the food industry.

Screening the biochemical composition of some marine microalgal species for feeding Cyclops abyssorum divergens

Recently, the use of microalgal bioproducts in aquaculture research has gotten consideration. The current study aims to screen the biochemical components of three microalgae: Nannochloropsis salina, Chlorella salina and Tetraselmis chuii for using in the feeding of Cyclops abyssorum divergens copepods that are suitable prey for the mouth gape of the cultured fish larvae. The effect of different initial densities of these algal cells used for copepods feeding was determined by estimation of proximate composition, polyunsaturated fatty acids (PUFAs), amino acids (AAs) and enzyme activities of copepods. N. salina showed high protein and lipid contents, while C. salina presented high carbohydrate content. C. salina and T. chuii presented high PUFAs, with proportions of 22.35 and 22.67%, respectively, and are considered as the source of n-3 fatty acids. Also, they had higher concentrations of total AAs (21.46 and 24.14 mg. g−1 dry wt., respectively). The experiment study showed that the C. salina diet at an initial concentration of 1 × 106 cells ml−1 resulted in a maximum increase of copepods population density (503.3%) and high protease and trypsin activities (42.65 and 31.80 U. mg−1 protein, respectively) in copepods. T. chuii diet at an initial concentration of 1 × 106 cells ml−1 resulted in a maximum protein, lipid, PUFAs and AAs contents of copepods. Therefore, C. abyssorum divergens copepods after 18 days feeding with an initial concentration of 1 × 106 C. salina or T. chuii cells ml−1 are the suitable candidate diets in larviculture because they achieved higher productivity and attained better nutritional composition of the copepods.

Binding loop of sunflower trypsin inhibitor 1 serves as a design motif for proteolysis-resistant antimicrobial peptides

Although antimicrobial peptides (AMPs) have become powerful drug candidates in the post-antibiotic era, but their low protease stability hinders their clinical application. In the present study, the natural sunflower trypsin inhibitor 1 (SFTI-1) binding loop (CTKSIPPIC) was used to design and synthesize a specific anti-proteolytic sequence template ((RX)n W (RX)n CTKSIPPIC (n = 2, 3; X represents A, I, L, V, F, and W)). After several antibacterial, bactericidal, and toxicity tests, RV3 stood out from the variants and had the highest average selectivity index (SI all = 156.03). It is highly stable in serum, varying pH, temperature, and salt ions as well as under high trypsin, pepsin, or papain concentrations. In a mouse skin inflammation model, established by Pseudomonas aeruginosa infection, RV3 could effectively kill the pathogen, promote wound healing, inhibit inflammatory cell infiltration, and inhibit mRNA and protein expression of TNF-α, IL-6, and IL-1β inflammatory factors. The antibacterial mechanisms of RV3 include combining with lipopolysaccharides and increasing cell membrane permeability, leading to cell membrane rupture and death. These findings indicate that RV3 has great potential for the treatment of bacterial infections.

Characterization of Seed Proteome Profile of Wild and Cultivated Chickpeas of India.

Chickpea is a widely grown legume in India, Australia, Canada, and Mediterranean regions. Seeds of chickpea are good source of protein for both human and animals. Wild relatives of chickpea (Cicer arietinum) are the potential gene pool for crop improvement; however, very little information is available on the seed proteome of these wild chickpeas. We aimed to analyze the seed proteome profiles of three wild relatives of chickpea, Cicer bijugum, Cicer judaicum and Cicer microphyllum along with two cultivated varieties JG11 and DCP 92/3. Total seed proteins were extracted using various extraction buffers for 2-D gel electrophoresis. Protein separated in a 2-D gels were subjected to image analyses, differentially expressed proteins were extracted from the gels and identified by the MALDI TOF/TOF. Seed protease inhibitors were analysed biochemically. We have standardized the 2-D gel electrophoresis method and separated seed proteins using the modified method. We identified a large number (400) of protein proteins which were differentially expressed in cultivated and wild type species of chickpea. A comparative analysis between C. bijugum and JG 11 revealed the presence of 9 over-expressed and 22 under-expressed proteins, while the comparison between C. bijugum with DCP 92/3 showed 8 over-expressed and 18 under-- expressed proteins. Similarly, comparative analysis between C. microphyllum with DCP 92/3 showed 8 over-expressed proteins along with 22 under-expressed proteins, while the comparative study of C. microphyllum with JG11 displayed 9 over-expressed and 24 under-expressed proteins. We also compared C. judaicum with DCP 92/3 which revealed 15 overexpressed and 11 under-expressed proteins. On the other hand, the comparative analysis of C. judaicum with JG11 showed 10 over-expressed proteins, while the numbers of under-expressed proteins were 14. Among the differentially expressed protein proteins, 19 proteins were analyzed by the MS/MS, and peptides were identified using the MASCOT search engine. In the wild relatives the differentially expressed proteins are phosphatidylinositol 4-phosphate 5- kinase, β-1-6 galactosyltransferase, RNA helicase, phenyl alanine ammonia lyase 2, flavone 3'-0-methyl transferase, Argonaute 2, Myb related protein, Tubulin beta-2 chain and others. The most important one was legumin having α- amylase inhibition activity which was up regulated in C. bijugum. We also studied the activity of protease inhibitor (trypsin and α- amylase inhibitors) in these seed lines which showed differential activity of protease inhibitors. The highest trypsin and α- amylase inhibition was observed in C. judaicum and C. bijugum, respectively. The differentially expressed proteins of wild relatives of chickpea appeared to be involved in various metabolic pathways. The study provides us information about the differences in the seed proteome of these wild species and cultivated varieties for the first time.