Enzymatic Digestion Research Articles

Isolation and identification of primary cells: A comprehensive primary cell culture experiment for graduate students.

Experimental teaching is an important part of postgraduate training in basic and clinical medicine. While primary cell isolation and identification are among the most important research techniques for medical graduate students, most graduate students do not understand and master these techniques before starting their research experience. In particular, many students lack training in this field, and high-quality teaching and learning materials are still very sparse. Here, we designed a practical experiment course for graduate students engaged in research. The target students usually have research projects involving primary cell culture in their future research, making the course highly applicable for the students. The lab exercise focused on the methods of primary cell isolation (including mechanical grinding method, explant culture method and enzymatic digestion method) and identification (including flow cytometry, immunofluorescence, and periodic acid-Schiff (PAS) staining). It aimed to help students master the conceptual, principle, technical, operation, and analytical skills related to primary cell culture and contributed to their foundation for future research. Students generally reflect that they have initially mastered the isolation and identification of primary cell culture as a result of the course. Student feedback also indicates significantly increased confidence in the practical application of primary cell culture in the future. Here, we provide our experience for others who may want to implement similar courses.

Acute physiological effects on macromolecule digestion following oral ingestion of the enzyme blend Elevase® in individuals that had undergone an ileostomy, but were otherwise healthy—a randomized, double blinded, placebo-controlled exploratory study

Digestive enzymes can selectively degrade proteins, carbohydrates and lipids; and their supplementation alongside food may accelerate the breakdown of complex food matrices, facilitate greater nutrient absorption, decrease food sensitivities and aid in the management of certain disease states. Several intrinsic and extrinsic factors govern food digestion and for every individual this phenomenon is unique. This study was conducted as a randomized, crossover, placebo-controlled design where each participant served as their own control. This post-hoc analysis investigated the impact of a dietary enzyme supplementation blend known as Elevase® on dietary macromolecule digestion in samples from otherwise healthy participants that had previously undergone a small bowel resection, resulting in an ileostomy (NCT04489810). This is the first time this study-paradigm has been used for the assessment of in vivo dietary breakdown following enzyme supplementation. Arguably, this technique offers superior data when compared to that generated in artificial gut digestion models, preclinical animal models, or indeed conventional clinical studies using stool analyses, as it allows real-time access to samples in situ in the small intestine where the majority of nutritional absorption takes place. It was demonstrated that after 4 h, Elevase® significantly increased monosaccharide levels (predominantly glucose and fructose) in the ileostomy samples taken from the same individuals on the same diet on a different day. In addition, the bile salt taurohyodeoxycholic acid was also increased, suggesting a physiological host response to the macromolecule digestion induced by the enzymatic blend. Overall, these findings suggest Elevase® could accelerate food digestion and potentially increase nutrient availability from the diet.

Performance of Laying Chickens with Feed Additive Cinnamon Flour (Cinnamomum burmanni (Nees & T.Nees) Blume)

Chickens are important commodities due to human need eggs to meet animal protein. Increasing of resistance of bacteria to synthetic antibiotics and consumer understanding of health and food safety have stimulated use of antibiotic growth promoters (AGP) in poultry industry. AGP applied sustainable feed management steps to improve gut health and poultry growth performance. Cinnamon, one of the spices as a natural product. This useful source due to antioxidants, anti-microbial, anti-inflammatory, antifungal, and hypo-cholesterolemic effects and its capability to activate digestion enzymes in intestine. This research aimed to analyze the performance of laying chicken eggs fed with cinnamon flour. The treatments used were cinnamon flour with levels R0 (basal ration, without cinnamon flour/CF, R1 (99.5% BR+ 0.5% CF), R2 (99% BR+ 1% CF), R3 (98% BR + 1.5 % CF), and R4 (98% BR + 2% CF). Data were analyzed with Completely Randomized Design and Duncan's test. Variables were ration consumption, Hen Day Production/HDP and ration conversion. Results showed that adding 2% cinnamon flour significant effect on feed consumption, HDP (P< 0,01) but insignificant on ration conversion (P>0.05). Cinnamon flour as feed additive source up to 2% level can increase the performance of laying hens.

High-Efficiency Degradation of PET Plastics by Glutathione S-Transferase under Mild Conditions.

Plastic pollution is a significant environmental concern globally. Plastics are normally considered chemically inert and resistant to biodegradation. Although many papers have reported enzyme-induced biodegradation of plastics, these studies are primarily limited to enzymes of microbial origin or engineered enzymes. This study reveals that poly(ethylene terephthalate) (PET, ∼6000 Da and 100 kDa) particles and plastic bottle debris (PBD, 24.9 kDa) can be efficiently degraded by a mammal-origin natural phase II metabolic isozyme, glutathione S-transferase (GST), under mild conditions. The degradation efficiency of PET plastics reached 98.9%, with a degradation rate of 2.6 g·L-1·h-1 under ambient or physiological conditions at 1 atm. PET plastics can be degraded by GST with varying environmental or biological factors (i.e., temperature, light irradiation, pH, and presence of humic acid or protein). We suggest a novel mechanism for PET degradation other than hydrolysis, i.e., the mechanism of cleavage and release of PET plastic monomers via nitridation and oxidation. This finding also reveals a novel function of GST, previously thought to only degrade small molecules (<1000 Da). This method has been successfully applied in real human serum samples. Additionally, we have tested and confirmed the ability to degrade PET of a mammal-origin natural digestive enzyme (trypsin) and a human-derived natural metabolic enzyme (CYP450). Overall, our findings provide a potential new route to plastic pollution control and contribute to our understanding of the metabolism and fate of plastics in organisms.

Potential bioaccessibility and bioavailability of polyphenols and functional properties of tiger nut beverage and its by-product during in vitro digestion.

"Horchata de chufa" is a beverage produced from tiger nut tubers, which yields a high amount of by-product. This study explored the functional properties of the Spanish tiger nut beverage (TNB) and its by-product (TNBP) together with the bioaccessibility and bioavailability of polyphenols in vitro. TNB and TNBP were characterized for polyphenols via LC/MS/MS and underwent in vitro digestion (INFOGEST). The total antioxidant capacity (TAC) of all bioaccessible fractions and digestion residues was assessed. Intestinal bioaccessible fractions were tested for the ability to inhibit the activity of digestive enzymes (α-amylase, α-glucosidase, and lipase) and the content of polyphenols, whose bioavailability was assessed in a Caco-2 cell model. Thirteen polyphenols were quantified and found to be more abundant in TNB (603 ± 1.4 μg g-1 DW) than in TNBP (187 ± 1.0 μg g-1 DW). Polyphenol bioaccessibility was higher for TNBP than that for TNB (57% vs. 27%), and despite a similar TAC of the intestinal bioaccessible fractions (10.2 ± 0.1 μmoL vs. 9.2 ± 0.03 μmoL eq. Trolox per g DW for TNB and TNBP, respectively), the different patterns of polyphenols released upon digestion suggested the higher ability of TNBP fraction to inhibit α-glucosidase and lipase. TNBP digestion residue showed higher TAC than TNB. Moreover, TNB polyphenols exhibited over 80% bioavailability, whereas TNBP polyphenols' bioavailability ranged from 62% to 84%. Overall, the findings demonstrated that TNBP maintains a high nutritional value, thus suggesting its possible reuse in innovative, healthy, and sustainable foods.

Wild-Edible Allium Species from Highlands of Eastern Anatolia: Phytochemical Composition and In Vitro Biological Activities

This study presents the phytochemical composition, antioxidant (hydrogen atom and single-atom transfer mechanisms), and digestive enzyme inhibitory (alpha-amylase, alpha-glucosidase, and pancreatic lipase) activities of ethanol-based extractions and traditional preparations (infusion) of the leaves of wild-edible Allium species (A. kharputense, A. affine, A. shirnakiense, and A. akaka) from the highlands of Eastern Anatolia. Among the eight extracts analyzed, ethanol extractions of the A. kharputense and A. akaka leaves exhibited better biotherapeutic activities and had the highest bioactive content. The dominant bioactive profile was composed of mainly allicin and phenolic compounds (chlorogenic acid, hesperidin, rutin, isoquercitrin, and quercetin) with small amounts of fatty acids. These data were similar to the biological activities and chemical composition of common Allium species and suggest the utilization of the extracts of wild-edible Allium species in the development of Allium-based biotherapeutics or nutraceuticals.

Prophylactic Effect and Antiulcerogenic Potential of Probiotic Lactiplantibacillus plantarum E1K2R2 and its Exopolysaccharide Against Ibuprofen-Induced Acute Gastric Ulcer.

Lactiplantibacillus plantarum (Lpb. plantarum), as a safe probiotic microorganism, has been documented for its production of multiple bioactive compounds, such as exopolysaccharides (EPS), which have been used in the treatment of many gastrointestinal diseases, including gastric ulcers. The present study aims to investigate the prophylactic and antiulcerogenic effects of the potential probiotic Lbp. plantarum E1K2R2 and its EPS against ibuprofen-induced gastric ulcer. A gastric ulcer model was established by feeding fasted rats with ibuprofen at a single dose (200 mg/kg body weight). The Lpb. plantarum E1K2R2 (109 CFU), its EPS (200 mg/kg bw), and the anti-ulcer reference drug (omeprazole) (20 mg/kg bw) were separately used to feed rats for seven consecutive days before ibuprofen administration. The mechanisms were meticulously examined, focusing on the anti-secretory activity and mucus production as well as the anti-inflammatory and antioxidant activities. The findings revealed that the gastro-preventive effect of Lbp. plantarum E1K2R2 (88.43%) was higher than that of the EPS (66.26%) and close to that of omeprazole (89.87%). This effect was achieved through similar mechanisms, including regulation of the secretory activity, augmentation of mucus production, mitigation of inflammation, and enhancement of the gastric mucosa's antioxidant capacity. Moreover, it was found that Lbp. plantarum E1K2R2 and its EPS induce the activities of gastric antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and S-transferase (GST); enhance glutathione (GSH) content; and reduce mucosal nitric oxide (NO), myeloperoxidase (MPO), and malondialdehyde (MDA) levels. Furthermore, histopathological and hematological examinations confirmed that both pre-treatments could effectively maintain the structural integrity of the gastric mucosa and improve some hematological parameters, respectively. This implies that Lpb. plantarum E1K2R2 and its EPS possess the potential to counteract ibuprofen-associated gastric ulcers, leveraging a variety of protective mechanisms.

Liposome-Papain Conjugates for Catalytic Digestion of Antibody Producing Fab Fragments.

Papain is useful for the enzymatic digestion of various proteins to produce functional peptides or protein fragments. Immobilized papain being reactive toward proteins and easily removable from a reaction mixture is worth developed. In the present work, liposomes were applied as colloidal carriers of papain for the catalytic digestion of polyclonal immunoglobulin G (IgG). Papain was covalently conjugated at pH = 7.0 via tris-succinimidyl aminotriacetate (TSAT) to liposomes incorporated with 5 mol % poly(ethylene glycol)-tethered lipid with a reactive amino group. The papain-conjugated liposome (liposome-papain) catalyzed the hydrolysis of Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride (BAPNA) at pH = 5.0-7.0. The activity of liposome-papain significantly increased with increasing temperature from 25 to 50 °C. The Michaelis constant Km was determined with respect to the liposome-papain- and free papain-catalyzed reactions with BAPNA at 37 °C as Km = 1.11 ± 0.13 and 11.6 ± 2.9 mM, respectively. Liposome-papain was applied to the catalytic digestion of 10 mg·mL-1 IgG at 37 °C for 24 h at pH = 5.0-7.0. The reaction mixture could be analyzed without pretreatment by using the affinity columns immobilized with the protein A or protein L ligand because colloidal liposome-papain quickly flowed through the chromatographic stationary phase, exhibiting little proteolytic effect on the proteinaceous ligands. The analysis clearly demonstrated the catalytic production of antigen-binding fragments (Fab) from IgG in an enzyme concentration- and pH-dependent manner. Liposome-papain with 15 or 50 mol % anionic lipids also catalyzed the formation of Fab from IgG. The above results demonstrated that liposome-papain was useful to digest IgG and to purify Fab formed with the affinity chromatography.

Nerve Regeneration Through Differentiation of Endometrial-Derived Mesenchymal Stem Cells into Nerve-Like Cells Using Polyacrylonitrile/Chitosan Conduit and Berberine in a Rat Sciatic Nerve Injury Model.

Nervous injuries are common in humans. One of the most advanced treatment methods is neural tissue engineering. This research aims to utilize nerve-like cells (NLCs) derived from endometrial mesenchymal stem cells (EnMSCs) on a polyacrylonitrile/chitosan (PAN/CS) scaffold, along with berberine, for the reconstruction of a rat sciatic nerve injury model. In this experimental study, EnMSCs were obtained through enzymatic digestion and identified using flow cytometry and their differentiation into adipocyte and osteoblast. PAN nanofiber scaffolds were produced through electrospinning, and EnMSCs were neurally differentiated on these scaffolds for grafting into an animal model. The expression of Nestin, Map-2, Tuj-1, and NF genes in NLCs was confirmed through RT-PCR and immunocytochemistry. Twenty-five adult male rats were used in this study, divided into 5 groups: (1) Scaffold/Cells/Berberine, (2) Scaffold/Cells, (3) Scaffold, (4) Berberine, and (5) Control. The animals were maintained for 8 weeks, and their sciatic nerve function (SFI) was assessed. Additionally, histological examinations were performed using hematoxylin/eosin, luxol fast blue staining, and immunohistochemistry. According to the results, extraction, identification, and differentiation of EnMSCs and fabrication of PAN conduit and its transplantation were successfully performed. The best behavioral performance and histology were observed in the Scaffold/Cells/Berberine group. The SFI test results were -24.08 for the Scaffold/Cells/Berberine group and -39.27 for the control group. The nerve diameter in these two groups was 591 µm and 80 µm, respectively, and the percentage of new nerve formation was 18.5% in the Scaffold/Cells/Berberine group and 0.2% in the control group. The immunohistochemistry results demonstrated that the intensity of the green color was higher in the groups with cells compared to the groups without cells. Furthermore, in the luxol staining results, all groups showed a significant improvement compared to the control group. In the Scaffold/Cells/Berberine group, fibers, and axons appeared denser, more organized, and displayed a higher intensity of blue staining. According to the results of this study, EnMSCs demonstrated efficient differentiation into NLCs. With the assistance of PAN/CS scaffolds and simultaneous administration of berberine, EnMSCs have the potential for nerve regeneration and recovery from sciatic nerve injury in the rat animal model.

Cholesterol-lowering effect of Pediococcus strains isolated from mother’s milk in mice

Purpose The Pediococcus strains belong to the group of lactic acid bacteria (LAB). This study aims to isolate and identify the Pediococcus spp. from mother’s milk, and investigate their tolerance to low pH and bile salts, antibacterial activity, attachment to HT-29 cells and effect on cholesterol-lowering and digestive enzymes in mice. Design/methodology/approach Pediococcus species were isolated from mother’s milk, then tested for tolerance to acid and bile salts, their antibacterial effect and attachment to HT-29 cell line. In mice experiment, the levels of triglyceride (TG), total cholesterol (TC), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C) and digestive enzymes such as amylase and lipase in serum of mice were measured after administration of 108 CFU/mL bacterial suspension. Findings A total of 24 Pediococcus spp. including 16 isolates of Pediococcus pentosaceus and 8 isolates of Pediococcus acidilactici were isolated. Fifteen isolates (62.5%) were able to survive in low pH and bile salt concentration. Ten isolates (41.6%) exhibited the most powerful inhibitory effects against both Gram-positive and Gram-negative bacterial pathogens. A total of four Pediococcus isolates (16.67%) showed strong attachment to HT-29 cells. In contrast to HDL-C and amylase, the serum levels of TC, TG and LDL-C were reduced and lipase was elevated significantly following administration of mother milk-derived Pediococcus isolates. Originality/value The Pediococcus isolates demonstrated good probiotic properties in particular cholesterol-lowering ability in mice. Thus, after more studies they can be considered as probiotic strain(s).

A closed-loop negative feedback model for the pancreas: A new paradigm and pathway to a cure.

To develop a model that describes how the pancreas functions, how the rate of synthesis of digestive enzymes is regulated, and finally what puts the pancreas to rest between meals. We applied the principals of control theory to previously published canine data to develop a model for how the canine pancreas functions. Using this model, we then describe the steps needed to apply this model to the human pancreas. This new closed-loop negative feedback model describes what regulates digestive enzyme synthesis. This model is based on basolateral exocytosis of butyrylcholinesterase (BCHE) into the interstitial space. It is this level of BCHE * BCHE activity that controls the rate of canine pancreas digestive enzyme synthesis, and in the absence of stimulation from the vagus nerve, puts the pancreas to rest between meals. Finding secretagogue-specific inhibitory enzymes in the human pancreas that are analogous to BCHE in the canine, and blocking its associated receptors, may lead to a cure for human pancreatitis.

Cross-linking affecting properties and in-vitro digestibility of starch-sucrose ester complexes

This study investigated the effects of cross-linking on the characteristics and in-vitro digestibility of starch-sucrose ester (SE) complexes. To achieve this, corn starch (CS) was cross-linked with various concentrations of sodium trimetaphosphate /sodium tripolyphosphate (5 %, 10 %, and 15 %). Subsequently, cross-linked starches (CLS) were complexed with SE through hydrothermal treatment. X-ray diffraction analysis revealed that V-type amylose-lipid complexes formed by the interaction between CS and SE. The resultant CS-SE complex significantly reduced CS digestibility, increasing its resistant starch (RS) content from 10.19 % to 22.71 %. The cross-linking modification did not alter the crystalline pattern of the CS-SE complex. Several CLS-SE complexes demonstrated higher enzymatic resistance compared to the CS-SE complex. The CLS10-SE complex exhibited the highest RS content of 39.37 % when the cross-linking agent concentration was 10 %. This phenomenon may be attributable to the cross-linking reaction having enhanced the interaction between starch molecular chains, reducing the solubility and swelling power, thereby hindering the accessibility of starch chains to digestive enzymes. These findings indicate that cross-linking modification is a practical approach to improving the anti-digestion performance of starch-lipid complexes.

Alleviative effects of C60 fullerene nanoparticles on arsenate transformation and toxicity to Danio rerio

Widely-used C60 fullerene nanoparticles (C60) result in their release into the aquatic environment, which may affect the distribution and toxicity of pollutants such as arsenic (As), to aquatic organism. In this study, arsenate (As(V)) accumulation, speciation and subcellular distribution was determined in Danio rerio (zebrafish) intestine, head and muscle tissues in the presence of C60. Meanwhile we compared how single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), graphene oxide (GO) and graphene (GN) nanoparticles alter the behaviors of As(V). Results showed that C60 significantly inhibited As accumulation and toxicity in D. rerio, due to a decrease in total As and monomethylarsonic acid (MMA) and As(V) species concentrations, a lower relative distribution in the metal-sensitive fraction (MSF). It was attributed that C60 may coat As(V) ion channels and consequently, affect the secretion of digestive enzymes in the gut, favoring As excretion and inhibiting As methylation. Similarly, MWCNTs reduced the species concentration of MMA and As(V) in the intestines, low GSH (glutathione) contents in the intestine. Due to the disparity of other carbon-based nanomaterial morphologies, SWCNTs, GO and GN exhibited the various effects on the toxicity of As(V). In addition, the possible pathway of arsenobetaine (AsB) biosynthesis included migration from the intestine to muscle in D. rerio, with the precursor of AsB likely to be 2-dimethylarsinylacetic acid (DMAA). The results of this study suggest that C60 is beneficial for controlling As(V) pollution and reducing the impact of As(V) biogeochemical cycles throughout the ecosystem.

Valorization of Lignocellulose with One-Step Acidified Monophasic Phenoxyethanol Fractionation.

Effective fractionation of lignocelluosic biomass and subsequent valorization of all three major components under mild conditions were achieved. Pretreatment with acidified monophasic phenoxyethanol (EPH) efficiently removed 92.6 % lignin and 80 % xylan from poplar at 110 °C in 60 min, yielding high-value EPH-xyloside, EPH-modified lignin (EPHL), and a solid residue nearly purely composed of carbohydrates. After removing the grafted acetyl groups using 1 % NaOH at 50 °C, the highest enzymatic digestibility reached 92.3 %. EPHL could be recovered in high yield and purity with an uncondensed structure, while xylose was converted to EPH-xyloside, a potential precursor in biomedical industries. Additionally, the acidified monophasic EPH solvent could effectively fractionate biomass from species other than hardwood, achieving over 70 % delignification from recalcitrant pinewood under the same mild conditions, demonstrating the high potential of monophasic EPH pretreatment.

Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Quenching Trypsin Is Unnecessary in Filter-Based Bottom-Up Proteomics.

Quenching digestions in proteomics prior to analysis is routine in order to eliminate residual protease activity. Residual activity leads to overdigestion, nonspecific star-activity, and back-exchange in isotopic 18O quantitation. Chemical and isobaric labeling (e.g., TMT/iTRAQ) of proteins or peptides for mass spectrometry-based proteomics is generally incompatible with ubiquitous postdigestion acidification. This necessitates buffer exchange and pH adjustments. We demonstrate that quenching is unnecessary with peptides generated from protein filter-traps, as trypsin activity and intact trypsin are negligible in the eluate from these preparations. Labeling can be directly performed on enzymatic digests from these methods, improving recovery, throughput, and ease of automation.

Integrated biofloc technology in red tilapia aquaculture: Salinity-dependent effects on water quality, parental stock physiology, reproduction, and immune responses

AbstractThe study examines the impact of integrated biofloc technology (BFT), different salinity levels, and their combined effects over 90 days on various physiological parameters. The investigation includes growth performance and feed utilization, water quality, the chemical composition of biofloc and fish, digestive enzymes, reproductive performance, stress and biochemical indicators, and antioxidant-immune responses in red tilapia (Oreochromis spp.) broodstock. The fish were initially weighed (males: n = 270; 104 ± 0.96 g; females: n = 270; 93.2 ± 0.66 g) and subsequently divided into 12 treatment groups (6 for males and 6 for females) spread across 36 separate tanks (3 tanks per treatment; 45 fish per treatment; 15 fish/tank). The treatments involved three salinity levels (18, 28, and 36 ppt) in both clear water (CW) and BFT systems. The outcomes demonstrated that fish in the 36 ppt salinity with BFT treatment demonstrated significant improvements (P &lt; 0.05) in growth parameters (final body weight, weight gain, and specific growth rate, feed intake, and feed conversion ratio). The condition factor in BFT groups increased in all salinity situations. The survival rates of broodstock were consistently high in all experimental conditions The study found that BFT and salinity significantly impacted (P &lt; 0.05) whole body contents (moisture, protein, lipid, and ash) in both males and females. Water quality parameters showed variations between BFT and CW, with notable impacts (P &lt; 0.05) on dissolved oxygen and pH. The BFT and salinity influenced digestive enzyme activities (protease, amylase, and lipase) and reproductive performance (males) and the 36 ppt salinity with BFT recorded the highest values. The hemato-biochemical and antioxidant-immune responses were also impacted by BFT and salinity exposure. The study highlights the potential benefits of incorporating BFT into red tilapia aquaculture systems, particularly in optimizing growth, health, and reproductive performance under various salinity conditions, which can enhance sustainable intensification, disease control, and environmental stewardship.

Spatiotemporal proteolytic susceptibility of allergens: positive or negative effects on the allergic sensitization?

From their expression in their respective allergenic source to their processing by antigen presenting cells, allergens continuously encounter proteases. The ability of allergens to resist to proteolysis by digestive enzymes or host-cell/microbial proteases is considered as an important property that influences their allergenic potential. However, the relationship between proteolytic stability and allergenicity is much more complex and depends on various factors, such as the protein structure dynamics, the exposure level, the route of sensitization, and their respective protease susceptibility. In this review, we summarize and discuss the current knowledge on several aspects of allergen proteolytic stability in different environments including the allergenic sources, routes of sensitization (skin, respiratory tract, gastrointestinal tract) and endolysosomal compartment of antigen-presenting cells. Proteolytic stability alone cannot represent a definitive criterion to allergenicity. The proteolytic susceptibility of allergens in processed extracts can affect allergy diagnosis and immunotherapy. Furthermore, the fine tuning of allergen stability during antigen processing can be exploited for the development of novel immunotherapeutic strategies.

Characterization of BrGH3A, a bovine rumen-derived glycoside hydrolase family 3 β-glucosidase with a permuted domain arrangement.

The bovine rumen contains a large consortium of residential microbes that release a variety of digestive enzymes for feed degradation. However, the utilization of these microbial enzymes is still limited because these rumen microorganisms are mostly anaerobes and are thus unculturable. Therefore, we applied a sequence-based metagenomic approach to identify a novel 2,445-bp glycoside hydrolase family 3 β-glucosidase gene known as BrGH3A from the metagenome of bovine ruminal fluid. BrGH3A β-glucosidase is a 92-kDa polypeptide composed of 814 amino acid residues. Unlike most glycoside hydrolases in the same family, BrGH3A exhibited a permuted domain arrangement consisting of an (α/β)6 sandwich domain, a fibronectin type III domain and a (β/α)8 barrel domain. BrGH3A exhibited greater catalytic efficiency toward laminaribiose than cellobiose. We proposed that BrGH3A is an exo-acting β-glucosidase from Spirochaetales bacteria that is possibly involved in the intracellular degradation of β-1,3-/1,4-mixed linkage glucans that are present in grass cell walls. BrGH3A exhibits rich diversity in rumen hydrolytic enzymes and may represent a member of a new clan with a permuted domain topology within the large family.

The effects of β-mannanase supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid production in broiler chickens fed diets with different metabolizable energy levels.

This study assessed the effects of β-mannanase (BM) supplementation on growth performance, digestive enzyme activity, cecal microbial communities, and short-chain fatty acid (SCFA) production in broiler chickens fed diets with different metabolizable energy (ME) levels. A total of 1,296 male one-d-old Cobb 500 broilers were randomly distributed in a 3 × 2 factorial arrangement (three ME levels × 0 or 200g/ton BM), with 6 replicates per treatment combination. The three ME levels were 3,000 (ME1), 2,930 (ME2), and 2,860 (ME3) kcal/kg, respectively, during the 0-3 w-old stages and 3,150 (ME1), 3,080 (ME2), and 3,010 (ME3) kcal/kg, respectively, during the 3-6 w-old stages. Reducing ME levels increased broiler feed intake (P = 0.036) and decreased average daily gain (ADG, P = 0.002) during the entire period. While BM supplementation increased ADG (P = 0.002) and improved the feed conversion ratio (P = 0.001) during the 0-3 w-old stages, with no effect during the 3-6 w-old stages. Overall, reducing ME levels increased pancreatic lipase (P = 0.045) and amylase (P = 0.013) activity and duodenal amylase activity (P = 0.047). Notably, BM supplementation significantly increased pancreatic lipase activity (P = 0.015) and increased lipase (P = 0.029) and amylase (P = 0.025) activities in the jejunal chyme. Although diet or enzyme supplementation did not affect microbial diversity, significant differences in microbial communities were observed. At the genus level, decreasing ME levels significantly affected the average abundances of Tyzzerella (P = 0.028), Candidatus_Bacilloplasma (P = 0.001), Vibrio (P = 0.005) and Anaerotruncus (P = 0.026) among groups, whereas BM supplementation reduced the average abundances of Escherichia-Shigella (P = 0.048) and increased the average abundances of Barnesiella (P = 0.047), Ruminococcus (P = 0.020), Alistipes (P = 0.050), and Lachnospiraceae_unclassified (P = 0.009). SCFA concentrations strongly depended on bacterial community composition, and BM supplementation increased acetic acid (P = 0.004), propionic acid (P = 0.016), and total SCFA concentrations. In conclusion, BM supplementation improved the performance of younger broilers, and both enzyme supplementation and reduced ME levels positively affected digestive enzyme activity and intestinal microflora.