RESUMO

Thermolysin (TLN) is a microbial highly-priced thermostable metallo-endoprotease with complementary substrate specificity to those of proteases widely used in science and industry for protein digestion and milk-clotting. This study is the first to immobilize TLN on aminated superparamagnetic nanoparticles (Fe3O4@silica-NH2) aiming for higher stability, recoverability, reusability, and applicability in proteolysis and as a microbial rennet-like milk-clotting enzyme. The nanobiocatalyst developed (Fe3O4@silica-TLN) displays hydrolytic activity on a synthetic TLN substrate and, apparently, was fully recovered from reaction media by magnetic decantation. More importantly, Fe3O4@silica-TLN retains TLN catalytic properties in the presence of calcium ions even after exposure to 60 °C for 48 h, storage at 4 °C for 80 days and room temperature for 42 days, use in proteolyses, and in milk-clotting for up to 11 cycles. Its proteolytic activity on bovine milk casein in 24 h furnished 84 peptides, of which 29 are potentially bioactive. Also, Fe3O4@silica-TLN catalyzed the digestion of bovine serum albumin. In conclusion, Fe3O4@silica-TLN showed to be a new, less autolytic, thermostable, non-toxic, magnetically-separable, and reusable nanobiocatalyst with highly attractive properties for both science (peptide/protein chemistry and structure, proteomic studies, and the search for new bioactive peptides) and food industry (cheese manufacture).

Assuntos

RESUMO

This study developed and evaluated chitosan-sodium alginate capsules containing the probiotic Lacticaseibacillus rhamnosus GG using extrusion and emulsification techniques. The encapsulated L. rhamnosus GG cells were also evaluated for technological and probiotic-related physiological functionalities, as well as when incorporated in UHT and powdered milk. Extrusion (86.01 ± 1.26%) and emulsification (74.43 ± 1.41%) encapsulation techniques showed high encapsulation efficiency and high survival rates of L. rhamnosus GG during 28 days of refrigeration and room temperature storage, especially emulsification capsules (> 81%). The encapsulated L. rhamnosus GG cells showed high survival rates during exposure to simulated gastrointestinal conditions (72.65 ± 1.09-114.15 ± 0.44%). L. rhamnosus GG encapsulated by extrusion and emulsification performed satisfactorily in probiotic-related physiological (pH and bile salts tolerance) and technological properties (positive proteolytic activity, diacetyl and exopolysaccharides production, high NaCl tolerance (> 91%), besides having high heat tolerance (> 76%)). L. rhamnosus GG in extrusion and emulsification capsules had high survival rates (> 89%) and did not significantly affect physicochemical parameters in Ultra-High Temperature (UHT) and powdered milk during storage. The results demonstrate that L. rhamnosus GG can be successfully encapsulated with alginate-chitosan as a protective material through extrusion and emulsification techniques. UHT and powdered milk could serve as appropriate delivery systems to increase the intake of this encapsulated probiotic by consumers.

RESUMO

In this study, the effect of in vitro gastrointestinal digestion of phenolic compounds, the total phenolic content, and the antioxidant potential of stingless bee honey were investigated. Among the 33 phenolic compounds investigated, 25 were quantified, and only eight were not bioaccessible (p-aminobenzoic acid, sinapic acid, pinobanksin, isorhamnetin, quercetin-3-glucoside, syringaldehyde, coumarin, and coniferaldehyde). Benzoic acid was predominant in most undigested samples (21.3 to 2414 µg 100 g-1), but its bioaccessibility varied widely (2.5 to 534%). Rutin, a glycosylated flavonoid, was quantified in all samples and might have been deglycosylated during digestion, increasing the bioaccessibility of quercetin in a few samples. Overall, the concentration of phenolic compounds prior digestion and their bioaccessibility varied greatly among samples. Nevertheless, higher concentrations before digestion were not correlated to greater bioaccessibility. This study is the first to assess the in vitro bioaccessibility of phenolic compounds in SBH, providing novel insights into SBH research.

Assuntos

RESUMO

Yeast and fibrolytic enzymes serve as additives incorporated into the nutrition of ruminants to regulate rumen fermentation and increase the digestibility of fiber, thereby enhancing the efficiency of rumen fermentation. Two experiments were conducted to assess the impact of five diets: a control diet without additives, diets with yeast (Saccharomyces cerevisiae) or exogenous fibrolytic enzymes (EFE), and diets with a blend of 0.7yeast + 0.3EFE or 0.7EFE + 0.3Yeast (based on recommended levels in g/kg of total DM). In the first experiment, 40 five-month-old Santa Ines lambs (mean weight 25.0 ± 1.3 kg) were distributed in a completely randomized design (5 treatments and 8 lambs) for 81 days to evaluate performance, ingestive behaviour, and serum metabolites. In the second experiment, 25 Santa Ines male lambs weighing 25.7 ± 4.1 kg were housed in metabolic cages, in a randomized design with 5 treatments and 5 lambs, evaluating digestibility, nitrogen balance, and rumen pH. EFE supplementation increased intakes of dry matter (DM), total digestible nutrients (TDN), and apNDF (mean of 38.1, 5.26, and 27%, respectively) compared to yeast or the 0.7yeast-0.3EFE blend. Feed conversion was most efficient (mean of 27.1%) in lambs fed Yeast, 0.7EFE + 0.3yeast, and the control diet. Lambs fed 0.7yeast + 0.3EFE spent less time eating (mean of 16.5%) and more time idling (mean of 10.75%), whereas EFE-fed lambs spent more time eating (mean of 19.73%), and 0.7EFE + 0.3yeast-fed lambs spent more time ruminating (mean of 20.14%). Control group lambs chewed and ruminated less (means of 24.64 and 17.21%, respectively) compared to other treatments. Lambs on the 0.7yeast + 0.3EFE blend had higher eating and rumination efficiency rates for DM and apNDF (mean of 19.11 and 17.95%, respectively) compared to other additive treatments or individual additives. They also exhibited lower (means 7.59 g/d) urinary N excretion, with improved N retention (mean 3185 g/d) compared to the control group. There were significant effects on serum albumin and cholesterol concentrations, with the 0.7yeast + 0.3EFE blend showing higher albumin (mean 4.08 g/dL) levels, while diets without additives and yeast-EFE blends had higher cholesterol (mean of 62.51 g/dL) concentrations. Including Saccharomyces cerevisiae yeast along with 0.7 yeast + 0.3 EFE blend is recommended when feeding similar lamb diets to those used herein because it improves the efficiency of intake, rumination of DM and NDF, and nitrogen utilization without affecting the lamb performance.

Assuntos

RESUMO

Vegetables and legume soups contain various essential and bioactive constituents such as vitamin C, carotenoids, and phenolics. Antioxidant activity characteristics related to those compounds are well known to contribute profusely to human health. The cooking technique affects the bioavailability of nutrients and bioactive compounds, making it crucial to explore optimal alternatives to maximize them. The objective of this study was to explore the influence of different cooking techniques (boiling, pressure cooking, sous-vide, and cook-vide) on the physicochemical properties and bioactive characteristics of a ready-to-eat vegetable lentil soup. For this, the bioaccessibility of those compounds was assessed through an in vitro simulated gastrointestinal methodology. The firmness of vegetables was established to define treatments' cooking times, allowing subsequent comparison of the nutritional and functional properties of the soups. The color of vegetables was also evaluated as a quality parameter, which contributed to providing a global vision of the process impact. The results revealed that in vitro digestion (IVD) caused a decrease in all bioactive compound determinations for all cooking treatments of up to 72% for total phenols, 92% for lycopene, 98% for carotenoids, and 100% for vitamin C. Additionally, the antioxidant activity of the soups after thermal treatment improved up to 46% measured by the DPPH method. This study emphasizes the importance of considering the digestion process in the selection of the most adequate cooking technique. After IVD, traditional cooking (boiling) reached the maximum total carotenoid and lycopene contents; cook-vide and pressure-cooking techniques provided the highest total phenol content, showing these three techniques to have the maximum antioxidant capacity.

RESUMO

Probiotics face harsh conditions during their transit through the gastrointestinal tract (GIT) of fish because of low-pH environments and intestine fluid. Therefore, the evaluation of probiotic viability under simulated gastrointestinal conditions is an important step to consider for probiotic supplementation in fish feed prior to in vivo trials. Therefore, this study aimed to evaluate the effect of stomach and intestinal simulated conditions on the viability of encapsulated Lactococcus lactis A12 using an in vitro digestion model for tilapia. A Box Behnken design was used to evaluate the potential effect of three factors, namely stomach pH, residence time in the stomach, and enzyme quantity, on the viability of encapsulated Lactococcus lactis A12. As the main results, low pH (4.00), long residence time (4 h), and enzyme quantity (2.68 U of total protease activity) led to lower final cell counts after the phases of the stomach and intestine. Encapsulated probiotic bacteria showed higher viability (p < 0.05) and antibacterial activity (p < 0.05) against the pathogen Streptococcus agalactiae than non-encapsulated bacteria. The results suggest that L. lactis A12 survives in GIT conditions and that the proposed in vitro model could be used to explore the viability of probiotic bacteria intended for fish feed supplementation.

RESUMO

Toxorhynchites mosquitoes have an exclusively phytophagous feeding habit as adults, which leads to significant differences in their morphophysiology compared with haematophagous mosquitoes. However, the molecular mechanisms of digestion in this mosquito are not well understood. In this study, RNA sequencing of the posterior midgut (PMG) of the mosquito Toxorhynchites theobaldi was undertaken, highlighting its significance in mosquito digestion. Subsequently, a comparison was made between the differential gene expression of the PMG and that of the anterior midgut. It was found that the most abundant proteases in the PMG were trypsin and chymotrypsin, and the level of gene expression for enzymes essential for digestion (such as serine protease, α-amylase and pancreatic triacylglycerol lipase) and innate immune response (including catalase, cecropin-A2 and superoxide dismutase) was like that of haematophagous mosquitoes. Peritrophin-1 was detected in the entire midgut, with an elevated expression level in the PMG. Based on our findings, it is hypothesized that a non-haematophagic habit might have been exhibited by the ancestor of Tx. theobaldi, and this trait may have been retained. This study represents a pioneering investigation at the molecular level of midgut contents in a non-haematophagous mosquito. The findings offer valuable insights into the evolutionary aspects of feeding habits in culicids.

Assuntos

RESUMO

According to the World Health Organization vector-borne diseases account for more than 17% of all infectious diseases, causing more than 700,000 deaths annually. Vectors are organisms that are able to transmit infectious pathogens between humans, or from animals to humans. Many of these vectors are hematophagous insects, which ingest the pathogen from an infected host during a blood meal, and later transmit it into a new host. Malaria, dengue, African trypanosomiasis, yellow fever, leishmaniasis, Chagas disease, and many others are examples of diseases transmitted by insects.Both the diet and the infection with pathogens trigger changes in many metabolic pathways, including lipid metabolism, compared to other insects. Blood contains mostly proteins and is very poor in lipids and carbohydrates. Thus, hematophagous insects attempt to efficiently digest and absorb diet lipids and also rely on a large de novo lipid biosynthesis based on utilization of proteins and carbohydrates as carbon source. Blood meal triggers essential physiological processes as molting, excretion, and oogenesis; therefore, lipid metabolism and utilization of lipid storage should be finely synchronized and regulated regarding that, in order to provide the necessary energy source for these events. Also, pathogens have evolved mechanisms to hijack essential lipids from the insect host by interfering in the biosynthesis, catabolism, and transport of lipids, which pose challenges to reproduction, survival, fitness, and other insect traits.In this chapter, we have tried to collect and highlight the current knowledge and recent discoveries on the metabolism of lipids in insect vectors of diseases related to the hematophagous diet and pathogen infection.

RESUMO

Antimicrobial resistance (AMR) is one of the main global health challenges. Anaerobic digestion (AD) can significantly reduce the burden of antibiotic resistance genes (ARGs) in animal manures. However, the reduction is often incomplete. The agronomic use of digestates requires assessments of their effects on soil ARGs. The objective of this study was to assess the effect of digestate on the abundance of ARGs and mobile genetic elements (MGEs) in the rhizosphere of ryegrass (Lolium perenne L.) and to determine whether half-dose replacement of digestate with urea (combined fertilizer) can be implemented as a safer approach while maintaining a similar biomass production. A greenhouse assay was conducted during 190 days under a completely randomized design with two experimental factors: fertilizer type (unfertilized control and fertilized treatments with equal N dose: digestate, urea and combined fertilizer) and sampling date (16 and 148 days after the last application). The results indicated that the digestate significantly increased the abundance of clinical class 1 integrons (intI1 gene) relative to the unfertilized control at both sampling dates (P < 0.05), while the combined fertilizer only increased them at the first sampling. Sixteen days after completing the fertilization scheme only the combined fertilizer and urea significantly increased the biomass production relative to the control (P < 0.05). Additionally, by the end of the assay, the combined fertilizer showed significantly lower levels of the macrolide-resistance gene ermB than digestate and a cumulative biomass similar to urea or digestate. Overall, the combined fertilizer can alleviate the burden of integrons and ermB while simultaneously improving biomass production.

Assuntos

RESUMO

Triacylglycerols (TAGs) are a primary energy source for marine mammals during lipid digestion. Walruses (Odobenus rosmarus divergens) consume prey with a high content of long-chain polyunsaturated fatty acids; however, their digestive physiology and lipid digestion remain poorly studied. The present study aims to model and characterize the gastric (PWGL) and pancreatic (PWPL) lipases of Pacific walruses using an in-silico approach. The confident 3D models of PWGL and PWPL were obtained via homology modeling and protein threading and displayed the structural features of lipases. Molecular docking analysis demonstrated substrate selectivity for long-chain TAG (Trieicosapentaenoin; TC20:5n-3) in PWGL and short-chain TAG (Trioctanoin; TC8:0) in PWPL. Molecular dynamics simulations demonstrate that PWGL bound to tridocosahexaenoin (TC22:6n-3), the protein is considerably stable at all three salinity conditions, but fluctuations are observed in the regions associated with catalytic sites and the lid, indicating the potential hydrolysis of the substrate. This is the first study to report on the digestion of TAGs in walruses, including modeling and lipases characterization and proposing a digestive tract for pinnipeds.

Assuntos