Enzymes Pepsin Research Articles

Functional Properties and Proximate Analysis of Fish Waste Protein Hydrolysate Processed Using Enzymes

A huge production of fish and their processing waste give rise to by-products comprises up to 70% depending on the species, size and processing method. The waste includes visceral parts, head, frames, bones, skin and cut-offs are rich source of protein with high functional properties. It is generally discarded which is a wastage of nutrient source and leading to environmental issues. Therefore, it was aimed to utilized the by-products for maximum recovery of nutrients by enzyme hydrolysis method for the preparation of protein hydrolysate with the used of papain and pepsin for digestion following different hydrolysis conditions. With the hydrolysis of papain enzyme (1 to 6%), the protein content of finfish waste protein hydrolysate ranges from 19.17% ± 0.06 to 73.14% ± 0.08 and that of shellfish waste protein hydrolysate prepared with 5,10 and 15% papain enzyme showed 26.73% ± 0.04 to 40.4% ± 0.5 which is comparatively low. Whereas the highest protein content was observed in 1% pepsin enzyme treated finfish waste protein hydrolysate with 80.55% ± 0.07. Besides, the hydrolysates were composed of 6.91% ± 0.05 to 10.46% ± 0.05 (moisture content), 0.6% ± 0.01 to 2.4% ± 0.01 (ash content) and 0.02% ± 0.005 to 0.09% ± 0.005 (fat content). The hydrolysates were highly soluble ranges from 72.73% ± 0.05 to 93.83% ± 0.1 which indicates development of small size hydrophilic with highly solvated polypeptide particles. A reduced phenomena of foaming capacity and stability were observed in shellfish waste protein hydrolysate in contrast with finfish waste protein hydrolysate. Similar pattern was also resulted in emulsifying stability index. Whereas the emulsifying activity index was in the range of 6.15 ± 0.03 to 9.85 ± 0.07 m2/g. The water holding capacity of finfish and shellfish waste protein hydrolysate ranges from 3.4 to 4.23 gm/gm hydrolysate and 1.53 to 1.63 gm/gm hydrolysate respectively which is resulted by the difference in molecular weight of the peptide. The hydrolysates extracted from different sample with different enzyme and concentration at varying conditions were more or less similar ranges from 3.7 to 4.1 gm/gm protein hydrolysate (oil holding capacity). Hence, high protein content with good functional properties of the protein hydrolysate prepared with the utilization of fish waste is a positive impact on the attempt made to recover nutrient by enzymatic hydrolysis.

Lysozyme Modification Using Proteolytic Enzymes.

The lysozyme in the chicken egg white consists of various bioactive amino acids. However, these compounds are inactive when they are in the sequence of parent proteins. They become active only when isolated from these proteins. The aim of this study was to modify lysozyme with proteolytic enzymes under specific conditions of the reaction environment so as to obtain active biopeptides. The physicochemical properties of the resulting preparations were also assessed. Our study showed that the modification of lysozyme with hydrolytic enzymes (pepsin and trypsin) under strictly specified conditions resulted in obtaining biopeptide preparations with new and valuable properties, as compared with native lysozyme. After the enzymatic modification of lysozyme, two structural fractions were distinguished in the composition of the resulting preparations-the monomeric fraction and the peptide fraction. The modified lysozyme exhibited high surface hydrophobicity and high total antibacterial activity despite the decrease in the hydrolytic activity. Modification of lysozyme with hydrolytic enzymes, especially pepsin, resulted in preparations with very good antioxidative properties.

A New Exopolysaccharide from a Wood-Decaying Fungus Spongipellis borealis for a Wide Range of Biotechnological Applications.

Fungi are a unique natural resource rich in polysaccharides, proteins, and other components. Polysaccharides are considered one of the most important bioactive components in fungi. Increasing numbers of studies have confirmed that fungal polysaccharides have various biological activities. Given these facts, the main aim of this investigation was to carry out isolation, identification, and structural characterisation of a new polysaccharide (EPS) derived from laboratory-cultured vegetative mycelium of a new Spongipellis borealis strain isolated from the environment. The examination of monosaccharides in the EPS demonstrated that the isolated biopolymer was composed mainly of glucose, galactose, and mannose monomers. The analysis of the methylation of the studied polymer indicated that it contained mainly terminal, →3)-linked, →4)-linked, and →2,4)-linked hexoses. The effect of fungal polysaccharides on S. borealis proteolytic enzymes (pepsin, trypsin, and pycnoporopepsin) and laccase activity was determined for the first time. Incubation of the enzyme preparation and EPS showed an influence of EPS on the stability of these enzymes, compared to the control values (without EPS).

Nutritional innovations in superior gilthead seabream (Sparus aurata) genotypes: Implications in the utilization of emerging new ingredients through the study of the patterns of secretion of digestive enzymes

To promote sustainability and efficiency for aquaculture production, there are two key actions: i) The implementation of novel nutritional strategies with more sustainable raw materials, that aim to reduce the dependence on fishmeal (FM) and fish oil (FO) in the feeds, and ii) the implementation of a successful breeding program addressed to improve fish growth, feed utilization, and health. Different studies performed on salmonids and European sea bass (Dicentrarchus labrax) support the existence of genetic variability for the utilization of plant or non-marine-based diets. Nevertheless, those studies do not consider that the better zootechnical performance obtained in selected fish can be associated with differences in digestive biochemistry. Therefore, the present study aimed to evaluate the changes in the levels of the activities of different digestive enzymes present in two populations of gilthead seabream (Sparus aurata): reference or selected for high growth. Furthermore, the effect of different diets including a variety of protein ingredients partially substituting FM (poultry meal, insect meal, and bacterial single-cell protein) was assessed on the digestive enzyme profile of both fish genotypes, after 90 days of feeding. Higher levels of certain protein-related enzymes (pepsin and chymotrypsin) were found in selected sea bream fish compared with reference fish. Selected fish also revealed higher ADCs for dietary amino acids, irrespective of the diet fed, compared with the reference group. These results, added to the better growth and feed utilization of selected sea bream compared with reference fish, suggest that selected fish are more able to utilize the combination of emergent ingredients for aquafeeds, showing changes in the pattern of digestive enzymes to face the different ingredients in diets, which indicates a higher plasticity of the digestive enzymes to face changes in dietary ingredients. Those changes could be reflecting a compensatory mechanism to improve the digestibility of the ingredient.

Bioactivities of Garcinia kola enzymatic hydrolysates at different enzyme–substrate ratios

Natural products, such as enzymatic hydrolysates and bioactive peptides from dietary sources, are safe alternatives to synthetic compounds linked to various deleterious effects. The purpose of this study is to determine the in vitro bioactivities (antioxidant and anti-inflammatory activities) of Garcinia kola seeds enzymatic hydrolysates (GKPHs) at different enzyme (pepsin)-substrate ratios. G. kola protein, isolated by alkaline solubilization and acid precipitation, was hydrolyzed with pepsin at varying enzyme–substrate (E:S) ratios. The antioxidant parameters investigated include 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging, hydrogen peroxide scavenging and ferrous ion (Fe2+) chelating activities. For anti-inflammatory properties, membrane stabilization and protein denaturation activities tests were used. GKPH produced at 1:32 had the highest degree of hydrolysis (66.27 ± 4.21%). All GKPHs had excellent in vitro anti-inflammatory properties. However, only enzymatic hydrolysates produced at 1:16 (E:S) ratio chelated iron (II) and as well had the highest percentage hemolysis inhibition of 84.45 ± 0.007%, percentage protein denaturation inhibition of 53.36 ± 0.01% at maximum concentration and exhibited highest DPPH scavenging activity (87.24 ± 0.10%). The enzymatic hydrolysates had excellent solubility, emulsifying and foaming properties. It could be deduced from this study that pepsin at a ratio of 1:16 of G. kola protein produced the most effective enzymatic hydrolysates in terms of their antioxidant and anti-inflammatory activities. G. kola pepsin enzymatic hydrolysates, thus, have potential in development as functional foods and as therapeutics pharmaceutical industries in the management of diseases associated with oxidative stress and inflammation owing to their excellent functional, antioxidant and anti-inflammatory properties.

Improving the functionality and biological properties of Iranian date palm (Phoenix dactylifera L) seeds protein with different proteases

In this study, date seeds as a major agricultural by-product were utilized for production of antioxidant peptides with techno-functional properties. Date seeds protein (DSP) was extracted and hydrolyzed by different enzymes (Alcalase, trypsin, pancreatin and pepsin). Then, the degree of hydrolysis (DH), amino acid composition, functional characteristics (e.g., solubility, and water- and oil-holding capacity), free radical inhibiting activity, reducing power, total antioxidant activity (TAA) and metal ion chelating activity of the hydrolysates (DSPHs) were examined. Chemical analysis of the obtained protein concentrate (∼ 65 %) indicated the presence of amide A and B regions. Also, amide I to III regions and secondary structures (α-helix, β-sheet, β-tern and random coils) were observed in the extracted DSP-protein. The DH and essential amino acids content of DSPHs were affected by enzyme type. The hydrolysis treatments resulted in a significant increase in the solubility of DSP, particularly in acidic pH levels and around its isoelectric point. In comparison to DSP, DSPHs also exhibited higher inhibitory activity against free radicals such as DPPH (72.5 %), ABTS (92.5 %), OH (64.5 %), and NO (57.9 %). Furthermore, DSPHs demonstrated greater reducing power (1.84), TAA (2.3), and enhanced chelating activity towards iron (73.2 %) and copper (40.1 %) transition ions. Various factors, including DH and the composition of amino acids, especially the amount of charged, hydrophobic, and antioxidant types, have played a crucial role in these outcomes. With their significant nutritive, functional, antioxidant, and metal ion chelating activities, DSPHs can serve as a natural additive to enhance the nutritional value and stability of various food products.

Preclinical trial of the effectiveness of a safety nasogastric tube to detect the tube position based on tidal volume and pepsin assay results in the gastrointestinal tract of Macaca fascicularis

BackgroundTube misplacement into the tracheobronchial tract is associated with pneumothorax in 0.5% of cases. NGT verification only detects the position of the tube at the end of the procedure. Therefore, a safe nasogastric tube (SNGT) was created to detect the NGT position in real time in a simple and inexpensive way. This study aimed to prove the effectiveness of the SNGT prototype in Macaca fascicularis.ResultAn SNGT producing 50% of the TV had 100% sensitivity and specificity in detecting the position of the tube at 100% of the TV, with a sensitivity of 100% and a specificity of 87.5%. There was a significant difference between the movement of the SNGT 50% TV and SNGT 100% TV airbags (p ≤ 0.05). However, there was no significant difference between the accuracy of placement of the 50% TV SNGT, 100% TV SNGT, and conventional NGT (p > 0.05). The pepsin enzyme had better sensitivity (100%) than pH paper (91.66%) in detecting the end-of-procedure tube position. This research has the potential to advance into human clinical trials.ConclusionSNGTs are highly effective in detecting the NGT position inside the respiratory and digestive tracts to prevent misplacement.

In Silico Analysis of Individual Fractions of Bovine Casein as Precursors of Bioactive Peptides—Influence of Post-Translational Modifications

Bovine casein is one of the most known precursors of bioactive peptides among food proteins. Thus far, in silico investigations addressing casein have taken no account of the impact of modifications of amino acid residues on the feasibility of bioactive peptide release. The present study aimed to determine the effect of such modification on the possibility of release of bioactive peptides from casein during simulated digestion. The αs1-, αs2-, β-, and κ-casein sequences were deposited in the BIOPEP-UWM protein database considering phosphorylated amino acids, cysteine residues forming disulfide bridges, and pyroglutamic acid residues. The frequency of occurrence of bioactive fragments and the frequency of their release by digestive enzymes were determined for the analyzed modified and unmodified proteins. Peptides found exclusively in the sequences of unmodified proteins were deemed as false-positive results. From 1.74% (β-casein A2) to 4.41% (αs2-casein B and D) of the false-positive results were obtained for the total frequency of occurrence of bioactive fragments (sums of frequencies computed for all activities). In turn, from 1.78% (κ-casein B) to 9.18% (β-casein A2 and A3) of false-positive results were obtained for the predicted total frequency of release of bioactive peptides by the system of digestive enzymes (pepsin, trypsin, and chymotrypsin).

Antioxidant and Pro-collagen Synthesis Activities of the Eriobotrya japonica Leaf Extract Using Enzyme

Purpose: We conducted a study that examined the physicochemical properties of Eriobotrya japonica leaf enzyme-assisted extract (EJLE) from Jeju Island.Methods: EJLE was prepared using the pepsin enzyme. The antioxidant and antiaging effects of EJLE were investigated to determine its utility as a functional material.Results: In the analysis of total amino acids, 16 kinds of components were isolated from EJLE. The DPPH and ABTS radical scavenging activities of EJLE were found to be 97.19±0.24% and 96.91±0.02% respectively in 500 μg/mL. Inhibition activities of elastase by EJLE were confirmed. In fibroblasts, EJLE at 500 μg/mL demonstrated a procollagen synthesis activity of approximately 52%.Conclusion: Our results confirmed that EJLE induced antioxidants in cosmetic materials and increased procollagen synthesis on the skin.

Effect of Purslane (Portulaca oleracea L.) on Intestinal Morphology, Digestion Activity and Microbiome of Chinese Pond Turtle (Mauremys reevesii) during Aeromonas hydrophila Infection.

Large-scale mortality due to Aeromonas hydrophila (A. hydrophila) infection has considerably decreased the yield of the Chinese pond turtle (Mauremys reevesii). Purslane is a naturally active substance with a wide range of pharmacological functions, but its antibacterial effect on Chinese pond turtles infected by A. hydrophila infection is still unknown. In this study, we investigated the effect of purslane on intestinal morphology, digestion activity, and microbiome of Chinese pond turtles during A. hydrophila infection. The results showed that purslane promoted epidermal neogenesis of the limbs and increased the survival and feeding rates of Chinese pond turtles during A. hydrophila infection. Histopathological observation and enzyme activity assay indicated that purslane improved the intestinal morphology and digestive enzyme (α-amylase, lipase and pepsin) activities of Chinese pond turtle during A. hydrophila infection. Microbiome analysis revealed that purslane increased the diversity of intestinal microbiota with a significant decrease in the proportion of potentially pathogenic bacteria (such as Citrobacter freundii, Eimeria praecox, and Salmonella enterica) and an increase in the abundance of probiotics (such as uncultured Lactobacillus). In conclusion, our study uncovers that purslane improves intestinal health to protect Chinese pond turtles against A. hydrophila infection.

Real-time monitoring of casein gel microstructure during simulated gastric digestion monitored by small-angle neutron scattering

The evolving structure of protein-based foods during the digestion process is critical to the release of nutrients. However, traditional in vitro monitoring of the gel micro- and nano-structure during digestion involves analysing sample aliquots taken at different digestion time periods. This can pose issues for some gels, such as casein-based gels, as they are sensitive to sample manipulation and environmental changes. Herein, a newly developed flow setup was utilised to monitor (at the micro- and nano-length scales) the gel protein network of rennet-induced (RG) and transglutaminase-induced acid gels (TG) in situ and in real-time during simulated gastric digestion using ultra-small and small-angle neutron scattering (USANS and SANS). The proteolysis kinetics of the gels were investigated at two different pepsin enzyme concentrations (2000 and 8000 U mL-1) and in two different solvent environments (H2O and D2O). Results indicate that the flowing in situ system had a greater effect on the microstructural breakdown of TG relative to the acid-sensitive RG, compared to the traditional static method. This is the first in situ digestion study observing the structural changes of large protein gel particles with USANS or SANS in real-time. Our findings advance the understanding of the kinetics of casein gel disintegration under simulated conditions of gastric digestion relating to pepsin enzyme concentration and solvent environment, and critically, the utilisation of a new in situ and real-time setup for neutron studies.

Potential of Dry-Cured Ham Bones as a Sustainable Source to Obtain Antioxidant and DPP-IV Inhibitory Extracts.

The utilization of animal bones as a protein source could be used as a sustainable pathway for the production of bioactive compounds. In this study, bones were pretreated with pepsin enzyme (PEP) and then sequentially hydrolyzed with Alcalase (PA) and Alcalase, as well as Protana prime (PAPP). The degree of hydrolysis, antioxidant activity, and DPP-IV inhibitory activity were measured. All three hydrolysates showed antioxidant and DPP-IV inhibitory activity; however, the highest result in both bioactivities was obtained with the PAPP hydrolysate. The obtained free amino acid content was 54.62, 88.12, and 668.46 mg/100 mL of hydrolyzed in PEP, PA, and PAPP, respectively. Pepsin pretreatment did not significantly affect the degree of hydrolysis; however, it is suggested that it promoted the cleavage of certain bonds for subsequent protease action. Accordingly, a total of 550 peptides were identified in PEP hydrolysate, 1087 in PA hydrolysate, and 1124 in PAPP hydrolysate using an LC-MS/MS approach. Pepsin pretreatment could be an effective method in the utilization of bone sources for the production of antioxidant and hypoglycemic peptides.

Physiochemical and Functional Properties of (Balanites Aeqyptiaca. Del) Hydrolysates by Pepsin and Pancreatin Proteases

Enzymatic hydrolysates from underutilized desert date could produce functional ingredient for desert region and population of the world. The current study investigated the physiochemical and functional properties of hydrolysates from Balanites aeqyptiaca using pancreatin, pepsin and combined pancreatin and pepsin enzymes. The hydrolysate obtained through the addition of the enzyme pancreatin, and pepsin combined gave (45.1%) yield. On the other hand, pancreatin hydrolysate gave maximum (45.9%) yield and pepsin hydrolysate was (37.4%) .The combined hydrolysate has maximum protein (80.58%),ash content (0.85%) mineral profile; Ca(0.12mg.100g),K(1.29mg/100g), Na(2.01mg/100g),Mg (1.09mg/100g),Fe( 0.23mg/100g), Cu (0.09mg/100g) , Zn( 0.26mg/100g) and excellent anti-nutrient alkaloid , oxalate ,phytate and saponin and total phenolic content of (0.084mgCat/g). Functional properties such as bulk density (1.02g/m) swelling index (3.45g/m) least gelation capacity (20.39%) of pancreatin hydrolysate and WAC (1.72g/g) and OAC (1.73g/g) from pepsin hydrolysate produced the best result than the combine hydrolysate. The maximum solubility (12%) at pH 9 was observed for all samples. The hydrolysate by combined enzymes had a high IVPD compared with pancreatin and pepsin hydrolysates. However, both showed a good general foaming and emulsifying properties. Foaming capacity of the pepsin hydrolysate samples increased with concentration at pH3 and pH7 while the foaming stability at pH7 and pH9 of combined hydrolysate was at 20 mg/ml and 40 mg/ml high comparable with pepsin and pancreatin, respectively. The emulsifying activity of combined hydrolysate was high at 10mg/ml and 50mg/ml at alkaline region. On the other hand, pepsin hydrolysate reveals excellent emulsifying activity (EAI) of 200 mg/m index (EAI) at pH 5. The hydrolysate obtained from pepsin decreased in emulsifying stability as concentration increased and pH shift to alkaline region. The hydrolysate by pepsin and pancreatin had better WHC, OHC and foaming properties due to their solubility however lower than the combined hydrolysate. The result indicates potential utilization of hydrolysate from Balanites aaqyptiaca. del for less protein rich cereal food formulations.

Nematode predatory ability of the fungus Duddingtonia flagrans affected by in vitro sequential exposure to ovine gastrointestinal tract

Duddingtonia flagrans is a nematophagous fungus employed as a biocontrol agent of gastrointestinal nematodes in ruminants. After oral ingestion and passage through the digestive tract of animals, this microorganism captures the nematodes in the feces. The drastic conditions of ruminant digestive tract could affect fungi chlamydospores and therefore biocontrol activity. The aim of this study was to evaluate in vitro the effect of four ruminant digestive segments on the concentration and nematode predatory ability of a Colombian native strain of D. flagrans. The sequential four-step methodology proposed evaluated conditions of the oral cavity, rumen, abomasum, and small intestine such as pH (2, 6, 8), enzymes (pepsin, pancreatin), temperature (39 °C), and anaerobiosis comparing short (7 h) and long (51 h) exposure times. The results showed that the nematode predatory ability of the fungi is affected by sequential exposure to gastrointestinal segments and this effect depends on the exposure time to those conditions. After short exposure (7 h) through the four ruminant digestive segments, the fungi had a nematode predatory ability of 62%, in contrast, after long exposure (51 h) the nematode predatory ability was lost (0%). Moreover, the number of broken chlamydospores was higher in the long-exposure assay.

Partial dietary fishmeal replacement with mixture of plant protein sources supplemented with exogenous enzymes modify growth performance, digestibility, intestinal morphology, haemato-biochemical and immune responses for Nile tilapia, Oreochromis niloticus

For the aquaculture sector, expanding the amount of plant proteins substituting fish meal (FM) is favorable. This study was conducted to evaluate the properties of diets containing a blend of plant protein sources (cotton seed meal, sunflower meal and jojoba (CSJ)) instead of FM and supplemented with a mixture of exogenous enzymes on the growth performance, intestinal morphology, blood indices, activity of endogenous and digestibility also, immune response of Nile tilapia, Oreochromis niloticus. Three groups of the experimental diet (29.5% crude protein) and (18.76 MJ kg −1 gross energy) were performed. The first group CSJ1 was (FM: CSJ = 2:1), the second group diet CSJ2 was (FM: CSM = 2:2) and the third CSJ3 was (FM: CSJ = 2:1), the replaced was based on content of protein. All groups of diets were supplemented with mixture of enzymes (Protease, pepsin, trypsin, xylanase and amylase) at 0 and 0.5 g kg −1 diet therefore six diet was formulated. Fingerlings (6.30 ± 0.09 g) were randomly allocated into eighteen plastic tank (450 L capacity) in triplicate per each treatment (25 fish/ treatment) for 90 days. After 90-days of the feeding trial, results indicated that the highest wight gain and best FCR, higher (P < 0.05) activity of trypsin and lipase, apparent digestibility coefficient of nutrients were detected in fish fed diet of FM: CSJ 2:1 and 2:2 supplemented with 0.5 g enzymes mixture kg−1 diet with insignificant differences between group. The diet incorporating FM: CSJ 2:1 with 0.5 g enzymes mixture showed the maximum (P < 0.05) villi length/width, ASA, and MSR values. Fish fed a diet containing FM: CSJ 1:2, and supplemented with 0.5 g of an enzyme mixture kg−1 diet had the maximum Hb, Htc, RBCs and WBCs values (P < 0.05). The highest (P < 0.05) TP and GL value was estimated in diet containing FM: CSJ; 1:2 without the addition of enzymes mixture. while, the best (P < 0.05) value of ALT and AST were showed in diet containing FM: CSJ 2:1 without the addition of enzymes. The highest TAC, GSH and SOD values were recorded by fish fed diet containing FM: CSJ 1:2 or 2:2 with 0.5 g enzymes mixture kg−1 diet. Fish fed diets comprising various ratios of FM: CSJ with 0.5 g enzymes mixture kg−1 diet had the lowest (P < 0.05) MDA value. In conclusion, using mixture of enzymes (protease, pepsin, trypsin, xylanase and amylase) at 0.5 g kg−1 in combination with a high blend of CSJ instead of FM; 2:2 improved the growth performance, digestibility, digestive enzyme, intestinal morphology, blood indices, and immune response of Nile tilapia.

Sequential ultrasound assisted deep eutectic solvent-based protein extraction from Sacha inchi meal biomass: towards circular bioeconomy.

The need for high-quality dietary proteins has risen over the years with improvements in the quality of life. Deep eutectic solvents (DESs) have been regarded as potential green alternatives to conventional organic solvents for protein extraction from press cake biomass, meeting the needs of sustainable development goals. Sacha inchi seed meal (SIM) is generated as a by-product of the seed oil extraction industries containing high protein content. The current study presents a novel ultrasound assisted DES method for the extraction of SIM protein in a sequential manner. Four different DESs were screened, out of which choline chloride (ChCl)/glycerol (1:2) gave promising results in protein recovery and was further selected. The sequential ultrasound-ChCl/glycerol could effectively extract high total crude protein content (77.43%) from SIM biomass compared to alone ultrasound (29.21%) or ChCl/glycerol (58.32%) treatment strategies. The SIM protein extracted from ultrasound-ChCl/glycerol exhibited high solubility (94.39%) at alkaline pH and highest in vitro digestibility (71.16%) by digestive enzymes (pepsin and trypsin). The protein characterization by SDS-PAGE and FTIR elucidated the structural properties and presence of different functional groups of SIM protein. Overall, the sequential ultrasound-ChCl/glycerol revealed its significant potential for one-step biorefining of the waste Sacha inchi meal biomass for circular bioeconomy.

Compound acidifier promoted the growth of hybrid grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) after high replacement of fish meal with cotton protein concentrate (CPC), improved stomach formation and digestibility, immune potency

Hybrid grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu) is a popular aquaculture species in recent years. The latest research has found that cottonseed protein concentrate (CPC) has been found to be an effective alternative to fishmeal in feed production, but high substitution rates could cause growth inhibition and organism damage in hybrid grouper. Compound acidifiers in previous studies could effectively enhance the growth and digestive performance of fish. Therefore, five isonitrogenous (50% crude protein) and isolipid (10% ether extract) diets were used: a reference diet (FM, 50% fishmeal) and four experimental diets (27.5% fishmeal, 27.27% CPC) containing acidifiers at 0%, 0.05%, 0.1%, and 0.2%, respectively. There were three replicates of 30 groupers per group and the experiment lasted for 8 weeks. The results showed a significant increase in WGR and SGR and a significant decrease in FCR with the addition of 0.05% acidifier (P < 0.05). The compound acidifier could attenuate the stomach growth stimulation induced by high CPC. The compound acidifier promoted enhanced activity of digestive enzymes Pepsin and AMS in the stomach (P < 0.05). It could down-regulate the expression of inflammatory cytokines and increase the expression of anti-inflammatory cytokines. In conclusion, the addition of compound acidifier effectively promoted the growth of the hybrid grouper with high CPC, It can effectively improve the stomach digestion and immune potency.

The fate of mamaku gum in the gut: effect on in vitro gastrointestinal function and colon fermentation by human faecal microbiota.

Mamaku is a fern indigenous to the Pacific Islands with a long history of use for therapeutic benefits such as to combat skin conditions and manage gastrointestinal discomfort; however, the scientific understanding is limited. In this study, we examined the effect of mamaku gum, extracted from different age fronds of the New Zealand Black tree fern (Cyathea medullaris, Mamaku) (stage 1: young, stage 2: fully grown and stage 3: old), on gut function using in vitro models of static digestion, enzyme activity and static colonic fermentation. Under simulated gastric and small intestinal conditions, mamaku polysaccharide (MP) was indigestible as there was no decrease in the molecular weight (Mw) of the polymer. Mamaku gum could reduce the activity of digestive enzymes (α-amylase, pepsin and lipase) in a concentration-dependent manner, with the stage 1 sample showing the highest inhibition and stage 3 the lowest. All three mamaku gum samples could also equally bind bile acids during intestinal digestion. During fermentation, human faecal microbiota utilised the mamaku gum and significantly increased the production of total short-chain fatty acids (SCFAs) and reduced the pH when compared with the blank. However, changes in SCFAs and pH for mamaku groups were less prominent than for inulin and guar gum control groups, suggesting lower fermentability of mamaku gum compared to the latter two. Furthermore, mamaku gum altered the composition of colonic microbiota, specifically reducing the ratio of Firmicutes to Bacteroidetes and increasing the relative abundance of Bacteroides, Enterococcus, Paraprevotella and Parabacteroides genera. No obvious difference between mamaku gum samples from stage 1, 2 and 3 was observed during fermentation. Collectively, these results suggest that mamaku gum may modulate the functionality of the host gut by reducing enzyme activity, binding bile acids, altering the colonic microbial composition and producing SCFAs.

Isolation, Characterization and Anti-Pepsin (HIV-Protease) Activity of Vernonia amygdalina Del and Cymbopogon citratus (de Condole) Stapf

The anti-pepsin enzyme (HIV-1) activities of crude extract of Vernonia amygdalina and Cymbopogon citratus were investigated using standard methods. A portion (0.5cm3) of pepsin enzyme solution was measured and transferred into viels, 2 cm3 of hemoglobin and 1 cm3 of the extracts were mixed together. The procedure was repeated for all the different extract. A standard control was also prepared using hemoglobin, enzyme and the various concentrations of the anti-retroviral drugs. A negative control was ran using only hemoglobin and pepsin enzyme whereas positive control was ran along with the standard using hemoglobin, enzyme and various concentrations of antiretroviral drugs. The mixture was allowed to incubate at 37 ºC, after 20 mins, 700 µl of 5% TCA was added to stop the reaction. It was then centrifuged at 14000 rev/min for 5 mins and the supernatant was collected. Absorbance was recorded spectrophotometrically at 280 nm. Each sample was taken in triplicate. UV-Visible spectrophotometer was used to detect the absorbance at 280 nm. The results of the anti-pepsin enzyme (HIV-1) activities showed that petroleum ether fraction of Cymbopogon citratus exhibited wavelength of maximum absorption (גmax) ranging from 0.788 nm - 0.789 nm and its ethyl acetate fraction exhibited גmax absorption ranging from 0.785 nm- 0.788 nm. Petroleum ether fraction of Vernonia amygdalina exhibited גmax absorption ranging from 1.383 nm-1.399 nm while its ethyl acetate fraction exhibited גmax absorption ranging from 1.384 nm-1.400 nm. The result of this work shows that both Cymbopogon citratus and Vernonia amygdalina ethyl acetate fraction have higher activity with Vernonia amygdalina being the highest. Column chromatography of the ethyl acetate fraction of Vernonia amygdalina led to the isolation of the compound which shows higher activity (1.384 nm- 1.400 nm). Available spectroscopic data using 1H NMR, 13C NMR and IR showed that the compound could be β-Sitosterol. The result of this work therefore showed that the extract from the leaves of Vernonia amygdalina could be used for the treatment of HIV-1(since it has the same sequence and family with the pepsin enzyme) and other diseases caused by targeted virus.

Inhibitory effects of Ephedra alte on IL-6, hybrid TLR4, TNF-α, IL-1β, and extracted TLR4 receptors: in silico molecular docking

Inflammation is a physiological reaction of the immune system required to remove the presence of pathogenic germs. Many herbal-derived extracts and phytoconstituents show anti-inflammatory effects. Among these natural phytoconstituents is Ephedra alte (E. alte), which shows pepsin enzyme inhibitory, antibacterial, and antioxidant activities. In this work, molecular docking study is conducted on five major human anti-inflammatory cytokines receptors (IL-6, hybrid TLR4, TNF-α, IL-1β, and extracted TLR4) to explore the molecular recognition process and complex ligand-receptor interactions of E. alte phytoconstituents. Human TLR4 receptor has been computationally extracted, for the first time, from the hybrid TLR4 human and VLRB inshore hagfish. Among E. alte phytoconstituents, only β-Sitosterol and Androstan-3-one have better LBE (Lowest Binding Energy) scores with inhibition constant (Ki) values than those of other tested compounds. The β-Sitosterol and Androstan-3-one results indicate that these compounds could be efficient inhibitors of inflammation and reduce the oxidative stress by interfering with the activity of the five studied proteins.