Evaluation of the Antimicrobial Activity of Triple Enzyme-Embedded Organic-Inorganic Hybrid Nanoflowers (hNFs) in Comparison with Powerful Antimicrobial Agent Chitosan.

Organic-inorganic hybrid nanoflowers (hNFs) have high stability, reusability, low production cost, and overcome substrate/product inhibition. Antimicrobial activity of various hNFs has been reported to overcome environmental microbial contaminations and infections, which are considered major public health problems. α-amylase, protease, and lipase are the most common industrial enzymes exerting antimicrobial activity; therefore, we synthesized triple enzyme (α-amylase, protease, and lipase)-embedded hNFs by using pancreatin to evaluate their antimicrobial activity in comparison with one of the most potent antimicrobial polymer chitosan. The broad spectrum of the antimicrobial properties of chitosan is used in industrial products, including cosmetics, food, agriculture, pharmaceuticals, and textiles. SEM analysis, thermogravimetric analysis (TGA), and the degree of deacetylation (%DD) were performed for chitosan characterization, where SEM, FTIR, EDX, and XRD analyses were performed for the characterization of hNFs. The catalytic activity of pancreatin and hNFs was evaluated by measuring lipase, α-amylase, and protease enzyme activities at 37 °C. Antibacterial activities of hNFs, pancreatin, and chitosan were tested on gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria, compared to the pancreatin and chitosan via agar and broth dilution methods. hNFs showed enhanced catalytic activity for protease, lipase, and α-amylase compared to pancreatin at different pH values (pH 8, 9). hNFs showed catalytic activity after being washed and reused up to six times, indicating their reusability and recoverability. hNFs showed significant antimicrobial activity, such as chitosan, Staphylococcus aureus, and Escherichia coli, compared to pancreatin. Our novel hNFs can be used to develop antimicrobial technologies to fight against environmental microbial contaminations and antibiotic resistance-driven environmental pathogens.

Exploring the α-amylase-inhibitory properties of tannin-rich extracts of Cytinus hypocistis on starch digestion.

Cytinus hypocistis(L.) L. is an edible parasitic plant that grows within the roots of its host. In addition to its use as famine food in the past, it is also tradidionally used for treating several illnesses such as intestinal problems, inflammations, tumors, and bleeding. This species is rich in hydrolysable tannins, compounds often associated with inhibiting starch digestion. Therefore, the present work investigated how effectively C. hypocistis tannin-rich extracts inhibited enzymes involved in starch digestion and if such effect also occurs in vivo. The latter premise was approached using the starch tolerance test in mice. Two optimized hydroethanolic extracts were used, a heat-assisted and an ultrasound-assisted extract, with known hydrolysable tannin content. Both extracts demonstrated potent inhibition of α-amylase. Inhibitions were of the mixed type with inhibitor constants in the 15 µg/mL range. The inhibition of the intestinal α-glucosidase was at least ten times less effective. The inhibition of the α-amylase was negatively affected by in vitro gastrointestinal digestion and bovine serum albumin. In vivo, both extracts inhibited starch digestion at doses between 100 and 400 mg/mL in healthy mice. The highest doses of the ultrasound and heat extracts diminished the peak glucose levels in the starch tolerance test by 46 and 59.3%, respectively. In streptozotocin diabetic mice, this inhibition occurred only at the dose of 400 mg/mL. Under this condition, diminution of the peak glucose concentration in the starch tolerance test was equal to 36.7% and 48.8% for the ultrasound and heat extracts, respectively. Maltose digestion was not inhibited by the C. hypocistis extracts. Qualitatively and quantitatively, thus, the actions of both extracts were similar. The results allow adding a new biological property to C. hypocistis, namely, the ability to decrease the hyper-glycemic excursion after a starch-rich meal, propitiating at the same time a diminished caloric intake.

Quantitative Imaging of the Action of vCPP2319, an Antimicrobial Peptide from a Viral Scaffold, against Staphylococcus aureus Biofilms of a Clinical Isolate.

The formation of biofilms is a common virulence factor that makes bacterial infections difficult to treat and a major human health problem. Biofilms are bacterial communities embedded in a self-produced matrix of extracellular polymeric substances (EPS). In this work, we show that vCPP2319, a polycationic peptide derived from the capsid protein of Torque teno douroucouli virus, is active against preformed Staphylococcus aureus biofilms produced by both a reference strain and a clinical strain isolated from a diabetic foot infection, mainly by the killing of biofilm-embedded bacteria. The direct effect of vCPP2319 on bacterial cells was imaged using atomic force and confocal laser scanning microscopy, showing that the peptide induces morphological changes in bacterial cells and membrane disruption. Importantly, vCPP2319 exhibits low toxicity toward human cells and high stability in human serum. Since vCPP2319 has a limited effect on the biofilm EPS matrix itself, we explored a combined effect with α-amylase (EC 3.2.1.1), an EPS matrix-degrading enzyme. In fact, α-amylase decreases the density of S. aureus biofilms by 2.5-fold. Nonetheless, quantitative analysis of bioimaging data shows that vCPP2319 partially restores biofilm compactness after digestion of the polysaccharides, probably due to electrostatic cross-bridging of the matrix nucleic acids, which explains why α-amylase fails to improve the antibacterial action of the peptide.

Silver nanoparticles affect wheat (Triticum aestivum L.) germination, seedling blight and yield.

The aim of the study was to evaluate the effect of two types of negatively charged quasi-spherical silver nanoparticles (AgNPs) at concentrations of 10, 20 and 30mgL-1 and silver ions at a concentration of 30mgL-1 on the growth, selected physiological aspects and yielding of wheat (Triticum aestivum L.) cv. Tybalt, and on plant resistance to seedling blight. Seed germination, α-amylase activity in seeds, morphology and infestation of seedlings by pathogens were assessed in a hydroponic treatment. Growth rate, PSII efficiency, heading and yield of the same plants were then analysed in pot culture. Results showed that the AgNPs and silver ions had a negative effect on roots, but reduced seedling blight and improved leaf area compared to the control. In addition, the AgNPs reduced with sodium borohydride in the presence of trisodium citrate at concentrations of 10 and 20mgL-1 stimulated germination, α-amylase activity and shoot length, which was not observed in the case of silver ions and the AgNPs reduced with sodium hypophosphite in the presence of sodium hexametaphosphate. In a pot experiment, the AgNPs improved plant growth, PSII efficiency, accelerated heading and increased yield-related parameters compared with the control. Results revealed the interaction strength in the following order: TCSB-AgNPs>SHSH-AgNPs>silver ions. TCSB-AgNPs in the lowest concentration had the most favourable effect, indicating their great potential for use in improving wheat cultivation.

Evaluation of antioxidant capacity and digestive enzyme activities in Mytilus galloprovincialis exposed to nanoplastics under different patterns of hypoxia.

In the marine environment, plastic pollution may occur simultaneously with hypoxia. However, current ecological risk assessments of nanoplastics have rarely considered the impact of additional environmental factors, such as hypoxia. In this study, we investigated the effect of polystyrene nanospheres (PS-NPs) on the digestive performance (antioxidant system and digestive enzymes) of mussels Mytilus galloprovincialis under different patterns of hypoxia (normoxia, constant hypoxia, and fluctuating hypoxia). The result showed that PS-NPs caused oxidative damage in the digestive glands of mussels, while all patterns of hypoxia exacerbated this oxidative damage. Activities of four digestive enzymes (α-amylase, cellulase, trypsin, and lipase) were examined. Among these, the activity of the α-amylase was inhibited by PS-NPs, and the inhibition was aggravated by all the hypoxia patterns. The cellulase activity and trypsin activity was enhanced by PS-NPs, and the increase was further stimulated by hypoxia. Lipase activity was not affected by PS-NPs alone, but significant inhibition was detected after the coexposure to PS-NPs and hypoxia. Conclusively, the combined stress of hypoxia and nanoplastics can significantly affect the digestive performance of mussels and may alter the mussel nutrient uptake strategy. Our work has provided new insight into the ecological risk assessment of plastics under global climate change.

Research Note: Corn energy and nutrient utilization by broilers as affected by geographic areas and carbohydrases.

Two experiments (Exp.) were conducted to evaluate the effects of exogenous carbohydrases on nutrient and energy utilization of corn with different compositions by broilers. In Exp. 1, a total of 448 Cobb 500 male chicks were distributed in a 2 × 4 factorial arrangement (corn from regions geographically located in the North or South of Brazil × 4 carbohydrases supplementation), with 8 replicate cages of 7 birds each. In Exp. 2, 672 Cobb 500 male chicks were fed 12 experimental feeds, in a 3 × 4 factorial arrangement [3 corn endosperm compositions (waxy, semi-dent, or semi-flint) × 4 carbohydrases], with 8 replicate cages of 7 birds. Birds were fed semi-purified test diets with 95.9% corn from d 14 to 24 in both studies. In Exp. 1, α-amylase, ß-xylanase, or carbohydrase complex (cellulase, glucanase, and xylanase) were added to the diet. In Exp. 2, α-amylase, ß-xylanase, or α-amylase + ß-xylanase were supplemented. Digestibility of DM, N, ether extract (EE), Ca, and P as well as AME, AMEn, and IDE were determined. In Exp. 2, jejunal starch digestibility was determined on d 24. Data were subjected to analysis of variance and means were compared by Tukey test (P ≤ 0.05). Corn from the North origin had higher AME, AMEn, and digestibility of DM and N compared to the South (P ≤ 0.05). Amylase supplementation led to increases of 3% in AME and 2% in N digestibility when compared to the non-supplemented feeds (P ≤ 0.01). In Exp. 2, the highest ME values and EE digestibility were observed in the semi-flint corn compared to the waxy, whereas the semi-dent presented the highest digestibility of N and starch. Corn diets supplemented with amylase + xylanase had improvements of 2.5% AMEn and 3% starch digestibility. In conclusion, energy and nutrient utilization of corn by broilers depend on the region where it was grown. Corn genetics, expressed by the endosperm composition, and carbohydrase supplementation influenced energy and nutrient utilization by broilers.

Physicochemical properties and bioactivity of polysaccharides from Isaria cicadae Miquel with different extraction processes: effects on gut microbiota and immune response in mice.

The effect of different extraction processes on the physicochemical characterization, digestibility, antioxidant activity and prebiotic activity of Isaria cicadae Miquel (ICM) fruiting body polysaccharides was studied. Furthermore, the effect of ultrasound-assisted extraction of ICM (U-ICM) on gut microbiota, the intestinal barrier and immune response was deeply explored. This study found that ICMs showed high indigestibility in both α-amylase and artificial gastric juice, indicating that ICMs have the potential as dietary fiber. In contrast, U-ICM had the best antioxidant activity and prebiotic potential. Meanwhile, there was a structure-activity relationship between the antioxidant activity of ICMs and the content of uronic acid, arabinose and galactose. When healthy mice were fed U-ICM for 42 days, the relative abundances of Lactobacillus, Akkermansia, and Bacteroides were found to increase significantly, while that of Clostridium decreased significantly. Meanwhile, U-ICM significantly promotes the expression of tight junction protein and the production of cytokines, indicating that U-ICM had the function of enhancing the intestinal barrier and regulating the host immune response. In conclusion, U-ICM as dietary fiber has the potential to be developed as a gut health-promoting prebiotic component or functional food. This research provided a valuable resource for further exploring the structure-activity relationship and prebiotic activity of ICMs.

The growth performance, antioxidant and immune responses, and disease resistance of Litopenaeus vannamei fed on diets supplemented with Indian ginseng (Withania somnifera).

In the current study, white-leg shrimp (Litopenaeus vannamei) were fed on diets containing varying doses of Withania somnifera aqueous extract (WSAE) at a rate of 0 (control), 0.5, 1.0, and 2.0 g/kg feed for 56 days. After the feeding trial, shrimps in all groups were challenged with the exposure to Vibrio harveyi for ten days during which animals' mortality was observed. It is noted that the dietary WSAE linearly and quadratically stimulated shrimp's growth indices particularly at the treatment of 2.0 g/kg feed. Compared to the control group, the WSAE-fed L. vannamei had significantly higher villi length, villi width, and absorption area particularly in the treatment of 2.0 g/kg feed. Furthermore, L. vannamei fed on WSAE-enriched diets consumed more feed and exhibited higher total proteolytic activity, lipase, and α-amylase activities as compared with the control group. The dietary WSAE at escalating levels linearly and quadratically enhanced the antioxidant activity (serum superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total antioxidant capacity, and reduced glutathione) and the immune response (total hemocyte counts, total protein, lysozyme, and phagocytic activity). Similarly, the mRNA expression levels of cMn-SOD, CAT, and GPx genes were linearly and quadratically upregulated in the hepatopancreas of L. vannamei fed on WSAE-enriched diets (especially in the 2.0 g/kg feed treatment), while their lowest levels were significantly observed in the control group. On the other hand, malondialdehyde levels were significantly decreased in WSAE-supplemented shrimp groups, and its highest levels were observed in animals fed on the control diet. After the bacterial exposure, the survival rates of L. vannamei fed on 1.0 and 2.0 g WSAE/kg feed (61.3% and 66.7%, respectively) were higher than those in the control animals. Taken together, the results obtained herein indicate that inclusion of WSAE in diets of L. vannamei effectively enhanced the growth, antioxidant biomarkers, immune response, and resistance to the V. harveyi infection, particularly at the treatment of 2.0 g/kg feed.

RNAi-mediated knockdown of gut receptor-like genes prohibitin and α-amylase altered the susceptibility of Galleria mellonella to Cry1AcF toxin.

BACKGROUND: Due to the prolonged usage of Bt-based biopesticides and Bt-transgenic crops worldwide, insects are continually developing resistance against Cry toxins. This resistance may occur if any mechanistic step in the insecticidal process is disrupted possibly because of the alteration in Cry-receptor binding affinity due to mutation in receptor genes. Compared to other lepidopteran insects, Cry receptor-related research has made asymmetric progress in the model insect Galleria mellonella. RESULTS: Present study describes the molecular characterization and functional analysis of five Cry toxin receptor-related genes (prohibitin, GLTP, α-amylase, ADAM and UDP-GT) and a gut repair gene (arylphorin) from the gut tissues of G. mellonella. Protein-protein docking analysis revealed that Cry1AcF putatively binds with all the five candidate proteins, suggesting their receptor-like function. These receptor-like genes were significantly overexpressed in the gut tissues of fourth-instar G. mellonella larvae upon early exposure to a sub-lethal dose of Cry1AcF toxin. However, targeted knockdown (by using bacterially-expressed dsRNAs) of these genes led to variable effect on insect susceptibility to Cry1AcF toxin. Insects pre-treated with prohibitin and α-amylase dsRNA exhibited significant reduction in Cry1AcF-induced mortality, suggesting their probable role as Cry receptor. By contrast, insects pre-treated with GLTP, ADAM and UDP-GT dsRNA exhibited no significant decline in mortality. This maybe explained by the possibility of RNAi feedback regulation (as few of the receptors belong to multigene family) or redundant role of GLTP, ADAM and UDP-GT in Cry intoxication process. CONCLUSION: Since the laboratory culture of G. mellonella develop Bt resistance quite rapidly, findings of the current investigation may provide some useful information for future Cry receptor-related research in the model insect.

Meta-analysis of the effects of the dietary application of exogenous alpha-amylase preparations on performance, nutrient digestibility, and rumen fermentation of lactating dairy cows.

Several studies have evaluated the effects of the dietary application of exogenous alpha-amylase preparations (AMA) as a strategy to increase total tract starch digestibility (TTSD) and milk yield (MY) in dairy cows, but the results have been inconsistent. Thus, the objective of this study was to evaluate the effects of the dietary application of AMA on the performance, digestibility, and rumen fermentation of lactating dairy cows using a meta-analytic method. A total of 18 peer-reviewed manuscripts (N = 32 treatment comparisons) from 2003 to 2019 were systematically identified following the PRISMA method. The weighted raw mean differences between dietary AMA and control treatments were compared with a robust variance estimation. Likewise, diet characteristics like crude protein (CP) content, NDF content, starch content, days in milk (DIM), experimental design (Latin square and continuous), and AMA dose (0 to 732 Kilo Novo units [KNU]/kg TMR) were used as covariates in a meta-regression, subgrouping, and dose-response analysis. Compared to the control, dietary AMA increased (P < 0.05) DM digestibility (69.32% vs. 68.30%), TTSD (94.62% vs. 94.10%), milk protein concentration and yield (3.11% vs. 3.08%; 1.14 vs. 1.10 kg/d) and tended to increase (P = 0.09) fat-corrected milk (35.96 vs. 35.10 kg/d), but no effects were observed on DM intake (22.99 vs. 22.90 kg/d) and feed efficiency (1.50 vs. 1.48). Dietary AMA tended (P = 0.10) to reduce rumen pH (6.27 vs. 6.30). Both the enzyme dose and DIM strongly influenced (P < 0.05) the effects of AMA on digestibility and performance. The dose-response analysis revealed that feeding 600 KNU/kg to high-producing early lactation (< 70 DIM) dairy cows increased FCM and milk protein. Accounting for the type of experimental design was associated with a lower between-studies-variance among comparisons. Overall, this meta-analysis supports the hypothesis that dietary AMA supplementation is associated with a better lactational performance in dairy cows. However, these effects are only suitable for high-producing early lactation dairy cows.

For more than a decade, starch-degrading enzymes (amylolytic enzymes) have been used as a strategy to increase total-tract starch degradation to increase milk yield of dairy cows. Therefore, we conducted a meta-analysis to evaluate the effectiveness of starch-degrading enzymes on starch digestion and milk yield in dairy cows. Collectively, results across the literature suggest that feeding starch-degrading enzymes increased the degradation of starch in the rumen of dairy cows and tended to increase milk yield. Our results suggest that starch-degrading enzymes could increase milk yield in high-producing early lactation dairy cows.

Incredible affinity of Kattosh with PPAR-γ receptors attenuates STZ-induced pancreas and kidney lesions evidenced in chemicobiological interactions.

Since ancient times, plants have been used as green bioresources to ensure a healthier life by recovering from different diseases. Kattosh (Lasia spinosa L. Thwaites) is a local plant with various traditional uses, especially for arthritis, constipation and coughs. This research investigated the effect of Kattosh stem extract (LSES) on streptozotocin-induced damage to the pancreas, kidney, and liver using in vitro, in vivo and in silico methods. In vitro phytochemical, antioxidative and anti-inflammatory effects of LSES were accomplished by established methods followed by antidiabetic actions in in vivo randomized controlled intervention in STZ-induced animal models for four weeks. In an in silico study, LSES phytocompounds interacted with antidiabetic receptors of peroxisome proliferator-activated receptor-gamma (PPAR, PDB ID: 3G9E), AMP-activated protein kinase (AMPK, PDB ID: 4CFH) and α-amylase enzyme (PDB ID: 1PPI) to verify the in vivo results. In addition, LSES showed promising in vitro antioxidative and anti-inflammatory effects. In contrast, it showed a decrease in weekly blood glucose level, normalized lipid profile, ameliorated liver and cardiac markers, managed serum AST and ALT levels, and increased glucose tolerance ability in the animal model study. Restoration of pancreatic and kidney damage was reflected by improving histopathological images. In ligand-receptor interaction, ethyl α-d-glucopyranoside of Kattosh showed the highest affinity for the α-amylase enzyme, PPAR, and AMPK receptors. Results demonstrate that the affinity of Kattosh phytocompounds potentially attenuates pancreatic and kidney lesions and could be approached as an alternative antidiabetic source with further clarification.

BIOFLAVONOIDS AS AGENTS FOR CORRECTING NITRO-OXIDATIVE STRESS AND SALIVARY GLAND FUNCTIONS IN RATS EXPOSED TO ALCOHOL DURING MODELED LIPOPOLYSACCHARIDE-INDUCED SYSTEMIC INFLAMMATORY RESPONSE.

OBJECTIVE: The aim: To investigate the effects of bioflavonoids (curcumin, epigallocatechin-3-gallate and quercetin) on nitro-oxidative stress and the functions of submandibular SGs in rats under alcohol exposure during SIR. PATIENTS AND METHODS: Materials and methods: The studies were conducted on 35 rats of the Wistar line weighing 205-220 g, divided into 5 groups of seven animals in each: the 1st group, control group I, included animals receiving isotonic sodium chloride solution intragastrically twice a day; the 2nd group, control group II, included rats exposed to alcohol (in a dose of 24 mg/kg intragastrically through gavage a twice a day) for last 2 weeks during lipopolysaccharide (LPS)-induced SIR; the rats of the 3rd, 4th and 5th groups exposed to alcohol during LPS-induced SIR, which also received bioflavonoids. The bioflavonoids ("Sigma-Aldrich, Inc.", USA) were as following: curcumin (in a daily dose of 200 mg/kg), epigallocatechin-3-gallate (in a daily dose of 40 mg/kg), and quercetin (in a daily dose of 200 mg/kg), respectively. SIR was induced by intraperitoneal administration of Salmonella typhi LPS (during the first week a dose of 0.4 µg/kg of body weight was administered 3 times a week; during the next 7 weeks of the experiment rats received 0.4 µg/kg of body weight once a week. The formation of superoxide anion radical (О2 -), activity of NO-synthase - total (NOS), its constitutive and inducible isoforms (cNOS, iNOS), and concentration of peroxynitrites and S-nitrosothiols were evaluated spectrophotometrically. To assess the functional status of submandibular SGs in their homogenate we determined α-amylase activity (spectrophotometrically) and the aquaporin-5 concentration (by enzyme-linked immunosorbent assay). through gav-age with orogastric cannul. RESULTS: Results: When applying bioflavonoids under the conditions of alcohol administration during SIR, NADH-induced .О2 - production decreased and yielded to the result in the control group II by 36.8% under administering curcumin, by 34.5% under administering epigallocatechin-3-gallate, and by 41.3% under administering quercetin. The total NOS activity in SGs tissues was inferior by 42.8% to the relevant data in the control group II (under curcumin administration), by 33.7% (under epigallocatechin-3-gallate administration) and by 46.6% (under quercetin administration); and the iNOS activity decreased by 47.0, 38.3 and 52.0%, respectively. Under the administration of bioflavonoids peroxynitrites concentration in the submandibular SGs tissues was inferior to the control group II by 35.6% (under curcumin administration), by 37.4% (under epigallocatechin-3-gallate administration), and by 39.3% (under quercetin administration); the content of S-nitrosothiols was lower by 34.5, 31.1 and 35.3%, respectively. The administration of bioflavonoids led to the changes in α-amylase activity in the submandibular SGs tissues: its values exceeded the relevant data in the control group II by 40.4% (under curcumin administration), by 38.2% (under epigallocatechin-3-gallate administration), and by 34.1% (under quercetin administration); under those conditions aquaporin-5 concentration grew in 2.66, 2.61 and 2.55 times, respectively. CONCLUSION: Conclusions: The use of bioflavonoids (curcumin, epigallocatechin-3-gallate, and quercetin) under the combined administration of 40% ethanol solution and LPS considerably limits the development of nitro-oxidative stress in the tissues of the submandibular SGs. The administration of the bioflavonoids increases the level of cNOS coupling, and improves the functional status of the submandibular SGs under the combined administration of alcohol and LPS enhancing the activity of α-amylase and concentration of aquaporin-5.

Screening of antimicrobial, antioxidant, antidiabetic activities, anatomical and morphological properties of Colchicum speciosum Steven (Colchicaceae).

Colchicum speciosum Steven species is a perennial stemless plant. C. speciosum is a flowering herb native to mountainous regions of northern Turkey, the Caucasus, and northern Iran. It has been known as "Vargit, Aci Çigdem, Güz Çigdemi". The present study reports the antimicrobial, antioxidant, α-amylase and α-glucosidase inhibitory activities of corm, leaf and flower methanol extracts, anatomical (light and electron microscopes) properties of root, corm, leaf, flowers and morphological characteristics of C. speciosum. Three different part of extracts C. speciosum were evaluated against E. coli ATCC 8739, S. aureus ATCC 6538, B. subtilis ATCC 19,659, C. albicans ATCC 10,231, C. krusei ATCC 14,243, and C. tropicalis ATCC 750. The most effective extract was found to be MeOH extract for corm and leaf against C. tropicalis ATCC 750 strain with MIC value 160 > µg/mL. It has been investigated first time anatomy of the tepal, ovary, anther, filament of C. speciosum. Leaf extract was the highest phenolic component (78.61842 µg GAE/ mg extract). As a result of DPPH• and ABTS•+ tests, it was determined that the leaf extract showed the best activity (IC50 = 6.568 µg/mL and IC50 = 3.243 µg/mL, respectively). Corm extract exhibited α-glucosidase inhibitory activity with an IC50 value of 21039 µg/mL. This is the first study of the in vitro antimicrobial, antioxidant, antidiabetic activities, detailed anatomical and morphological properties of C. speciosum. HiGHLiGHTS : • Antioxidant-antidiabetic-antimicrobial potential of Colchicum speciosum • Leaf extract had the highest phenolic component • The leaf got the highest DPPH• and ABTS•+ antioxidant potential • Corm extract exhibited α-glucosidase inhibitory activity • The most effective extract was found to be MeOH extract for corm and leaf against C. tropicalis • This is the first study of the in vitro antimicrobial, antioxidant, antidiabetic activities, detailed anatomical and morphological properties of C. speciosum.

Impact of feed supplement with alfa-amylase and beta-glucanase on ingestive-related biomarkers registered with real-time sensors.

The aim of this study was to investigate the impact of feed supplements with alfa-amylase and beta-glucanase (Optipartum C+ 200) on ingestive-related behaviour biomarkers registered with real-time sensors: rumination behaviours and reticulorumen parameters (pH and temperature). Cows (n=20) in the treatment group (TG) were fed with Optipartum C+ 200 (Enzymes feed supplement: Alfa-Amylase 57 Units; Beta-Glucanase 107 Units) from 21 days before calving until 30 days after calving with a feeding rate of 200 g/cow/day. Cows (n=22) in the control group (CG) were fed a feed ration without feed supplement. Measurements started from 6 days before calving and continued until 21 days after calving. The following indicators were registered: with the RumiWatch System: Rumination time; Eating time; Drinking time; Rumination chews; Eating chews; Drinking gulps; Bolus; Chews per minute; Chews per bolus. With the SmaXtec system: the temperature, pH of the contents of the cows' reticulorumens, and cows' walking activity. According to our results, feed supplementation with alfa-amylase and beta-glucanase (Optipartum C+ 200) in the TG group resulted in increases in the following parameters: 9% rumination time and eating time, 19% drinking time, 11% rumination chews, 16% eating chews, 13% number of boluses per rumination, 5% chews per minute and 16% chews per bolus. The rumination time showed a strong, positive relation with rumination chews and bolus indicators in both groups (TG and CG) (p⟨0.001); while the rumination time in both groups of cows showed an opposite direction and was negatively related to eating time and eating chews (p⟨0.05). We found a 1.28 % lower reticulorumen pH and a 0.64 % lower reticulorumen temperature in cows fed with the supplement compared with cows in the control group. Cows in TG were 8.80% more active than those in the CG group. For improvement of ingestive-related behaviour we suggest adding a feed supplement with alfa-amylase and beta-glucanase (Optipartum C+ 200).

Strain variation in Bacillus cereus biofilms and their susceptibility to extracellular matrix-degrading enzymes.

Bacillus cereus is a foodborne pathogen and can form biofilms on food contact surfaces, which causes food hygiene problems. While it is necessary to understand strain-dependent variation to effectively control these biofilms, strain-to-strain variation in the structure of B. cereus biofilms is poorly understood. In this study, B. cereus strains from tatsoi (BC4, BC10, and BC72) and the ATCC 10987 reference strain were incubated at 30°C to form biofilms in the presence of the extracellular matrix-degrading enzymes DNase I, proteinase K, dispase II, cellulase, amyloglucosidase, and α-amylase to assess the susceptibility to these enzymes. The four strains exhibited four different patterns in terms of biofilm susceptibility to the enzymes as well as morphology of surface-attached biofilms or suspended cell aggregates. DNase I inhibited the biofilm formation of strains ATCC 10987 and BC4 but not of strains BC10 and BC72. This result suggests that some strains may not have extracellular DNA, or their extracellular DNA may be protected in their biofilms. In addition, the strains exhibited different patterns of susceptibility to protein- and carbohydrate-degrading enzymes. While other strains were resistant, strains ATCC 10987 and BC4 were susceptible to cellulase, suggesting that cellulose or its similar polysaccharides may exist and play an essential role in their biofilm formation. Our compositional and imaging analyses of strains ATCC 10987 and BC4 suggested that the physicochemical properties of their biofilms are distinct, as calculated by the carbohydrate to protein ratio. Taken together, our study suggests that the extracellular matrix of B. cereus biofilms may be highly diverse and provides insight into the diverse mechanisms of biofilm formation among B. cereus strains.

Antibiofilm Activity of α-Amylase from Bacillus subtilis and Prediction of the Optimized Conditions for Biofilm Removal by Response Surface Methodology (RSM) and Artificial Neural Network (ANN).

α-amylase is known to have antibiofilm activity against biofilms of both Gram positive and Gram-negative bacterial strains. Partially purified α-amylase from Bacillus subtilis was found to have inhibit biofilm formed by P. aeruginosa and S. aureus. The spectrophotometric and microscopic studies revealed that the antibiofilm efficacy of the working strain is greater than commercially purchased α-amylase. Response surface methodology (RSM) and artificial neural network (ANN) help to predict the optimum conditions [pH 8, treatment time 6 h and enzyme concentration (200 µg/mL)] for maximum biofilm eradication. This was confirmed by several in vitro experiments. Molecular docking interactions of α-amylase with the extracellular polymeric substances (EPS) of both P. aeruginosa and S. aureus indicate towards the existence of an efficient energy driven spontaneous process. Thus, this study highlights a combination of experimental and computational approach showing the naturally extracted α-amylase from B. subtilis having the potency of removing the biofilms of harmful bacterial strains involved in causing various nosocomial infections.

Growth phase and dietary α-amylase supplementation effects on nutrient digestibility and feedback enzyme secretion in broiler chickens.

Growth performance, nutrient digestibility, intestinal health, and endogenous enzyme secretion responses to dietary α-amylase supplementation during 4 growth phases of broiler chickens fed corn-soybean meal-based diets were evaluated in the present study. A total of 1,136 male broiler chicks were assigned at day 0 after hatching to 8 treatments in a 2 × 4 factorial arrangement. There were 2 dietary levels of α-amylase supplementation of 0 or 80 kilo-Novo alpha amylase units per kg diet and 4 posthatching growth phases of day 0 to 11, day 11 to 21, day 21 to 42, or day 42 to 56 in a randomized complete block design. Each treatment comprised 8 replicate pens, with either 25 (day 0-11), 20 (day 11-21), 16 (day 21-42), or 10 (day 42-56) birds per pen. Body weight gain and feed efficiency of birds improved (P < 0.01) with α-amylase supplementation. There were main effects of α-amylase, growth phase, and interaction (P < 0.01) on apparent ileal digestibility (AID) of starch. This ranged from 0.8% during day 11 to 21 to 2.8% during day 0 to 11 after hatching. The total tract retention of starch increased (P < 0.05) with amylase supplementation but was not different across growth phases. Amylase supplementation increased (P < 0.05) AID of gross energy, AME (kcal/kg), and AMEn (kcal/kg). Villus height in the jejunal tissue was increased (P < 0.01) by α-amylase supplementation. During day 11 to 21 after hatching, the viscosity of jejunal digesta and pancreatic amylase activity increased (P < 0.01) with amylase supplementation. In conclusion, dietary amylase supplementation improved growth performance, apparent nutrient digestibility, and digestive enzyme activity of broiler chickens fed a corn-soybean diet. The study indicates that the growth phase of birds may affect response to exogenous amylase.

Intestinal starch and energy digestibility in broiler chickens fed diets supplemented with α-amylase.

Dietary starch is the major energy source for broiler chickens; therefore, relevant information on its intestinal utilization is important. The present study was designed to evaluate intestinal starch and energy digestibility of broiler chickens fed diets supplemented with α-amylase. A total of 240 day-0 male broiler chicks were randomly assigned to 3 nutritionally adequate corn-soybean-based experimental diets comprising 3 levels of α-amylase supplementation (0, 80, or 160 KNU/kg diet). Each treatment comprised 8 replicate cages of 10 birds each. At day 21 after hatching, digesta was collected from 4 intestinal sites: the anterior jejunum (AJ), posterior jejunum (PJ), anterior ileum (AI), and posterior ileum. Increasing α-amylase supplementation linearly improved (P < 0.01) overall BW gain and feed efficiency of the birds. There were linear and quadratic (P < 0.01) responses of increasing α-amylase supplementation on starch and energy digestibility at the PJ and AI. The total tract digestibility of starch increased (P < 0.05) with increasing α-amylase supplementation. Starch disappearance and digestible energy (kcal/kg) linearly increased (P < 0.01) with digesta flow from the AJ to PJ as dietary α-amylase supplementation increased. There were linear (P < 0.01) and quadratic (P < 0.05) effects of increasing α-amylase supplementation on the villus height in the jejunum. The viscosity of the jejunal digesta decreased (P < 0.05) with increasing dietary α-amylase supplementation. The results from this study showed the efficacy of exogenous amylase in improving growth performance and starch and energy digestibility in broiler chickens. Furthermore, the digestibility of starch and energy and the impact of the exogenous amylase were higher at the PJ than other intestinal sites.

[Anticancer effect of modified banana (Musa cavendish AAA) starch in rats with 1.2-dimethylhydrazine]. / Efecto anticancerígeno del almidón modificado de banano (Musa cavendish AAA) en ratas con 1,2-dimetilhidrazina.

INTRODUCTION: Introduction: resistant starch (RS) is not completely digested in the human intestine but is fermented in the colon; intestinal pH decreases as short-chain fatty acids are produced. This is beneficial for health, and for preventing and treating rectal colon cancer. Pyrodextrinization and enzymatic hydrolysis are modifications to native starch (NS) that may increase the amount of RS. Objective: the objective of this project was to evaluate the effects of M. cavendish AAA native and both chemically and enzymatically modified starches on tumor markers in rats. Methods: modifications (chemical and enzymatic) were made to M. cavendish AAA NS, and were evaluated in rats with 1,2-DMH. Male Sprague Dawley rats (25) were used, divided into five experimental groups: PC, NC, NS, PI, and ERM. During 4 weeks they received the experimental diet assigned to each group. The PC, NS, PI and ERM groups received 2 weekly s.c. (subcutaneous) injections of 1,2-DMH (40 mg/kg) (third and fourth week). In feces, pH, ß-glucuronidase enzyme, and short-chain fatty acids were evaluated, and a histopathological study was performed of the intestine to detect microscopic lesions. Results: the activity of ß-glucuronidase decreased (p < 0.05) for NS, PI and ERM vs. PC. The highest proportion of butyric acid was observed in the NS (p < 0.05) vs. NC group. Sixty percent of enteritides were severe in grade in the PC group, and 40 % in the experimental groups. Conclusions: native starch granules resisted pyrodextrinization, but treatment with α-amylase broke the structure of the pyrodextrin granule. According to the treatments given to the rats, as the amount of RS present in the diet increases (NS), the neoplastic cells do not advance beyond the basement membrane, suggesting a possible cell-protective or anticancer effect.

INTRODUCCIÓN: Introducción: el almidón resistente (AR) no se digiere completamente en el intestino humano sino que se fermenta en colon; disminuye el pH intestinal, ya que se producen ácidos grasos de cadena corta, interviniendo de manera benéfica en el tratamiento preventivo y curativo del cáncer de colon rectal. La pirodextrinización y la hidrólisis enzimática son modificaciones al almidón nativo (AN) que pueden incrementar la cantidad de AR. Objetivo: el objetivo de este proyecto fue evaluar los efectos del almidón nativo de M. cavendish AAA y de los almidones modificados química y enzimáticamente sobre diversos marcadores tumorales en ratas. Métodos: se realizaron modificaciones (química y enzimática) del AN del banano M. cavendish AAA y se evaluaron en ratas tratadas con 1,2-DMH. Se utilizaron 25 ratas Sprague Dawley machos divididas en cinco grupos experimentales: CP, CN, AN, PI y MER. Durante 4 semanas recibieron la dieta experimental asignada a cada grupo. Los grupos CP, AN, PI y MER recibieron 2 inyecciones s.c. (subcutáneas) semanales de 1,2-DMH (40 mg/kg) (semanas 3 y 4). En las heces se evaluaron el pH, la enzima ß-glucuronidasa y los ácidos grasos de cadena corta, y se realizó un estudio histopatológico del ciego y el colon para detectar lesiones microscópicas. Resultados: la actividad de ß-glucuronidasa disminuyó (p < 0,05) para los grupos AN, PI y MER en comparación con el CP. La mayor proporción de ácido butírico se observó en el AN (p < 0,05) frente al CN. El 60 % de las enteritis fueron de grado severo en el CP, mientras que en los grupos experimentales fueron de 40 %. Conclusiones: los gránulos de almidón nativo resistieron la pirodextrinización pero el tratamiento con α-amilasa rompió la estructura del gránulo de pirodextrina. De acuerdo a los tratamientos suministrados a las ratas, conforme mayor es la cantidad de AR presente en la dieta (AN), las células neoplásicas no avanzan más allá de la membrana basal, sugiriendo un posible efecto protector o anticancerígeno celular.

Biofilm-Forming Ability and Effect of Sanitation Agents on Biofilm-Control of Thermophile Geobacillus sp. D413 and Geobacillus toebii E134.

Geobacillus sp. D413 and Geobacillus toebii E134 are aerobic, non-pathogenic, endospore-forming, obligately thermophilic bacilli. Gram-positive thermophilic bacilli can produce heat-resistant spores. The bacteria are indicator organisms for assessing the manufacturing process's hygiene and are capable of forming biofilms on surfaces used in industrial sectors. The present study aimed to determine the biofilm-forming properties of Geobacillus isolates and how to eliminate this formation with sanitation agents. According to the results, extracellular DNA (eDNA) was interestingly not affected by the DNase I, RNase A, and proteinase K. However, the genomic DNA (gDNA) was degraded by only DNase I. It seemed that the eDNA had resistance to DNase I when purified. It is considered that the enzymes could not reach the target eDNA. Moreover, the eDNA resistance may result from the conserved folded structure of eDNA after purification. Another assumption is that the eDNA might be protected by other extracellular polymeric substances (EPS) and/or extracellular membrane vesicles (EVs) structures. On the contrary, DNase I reduced unpurified eDNA (mature biofilms). Biofilm formation on surfaces used in industrial areas was investigated in this work: the D413 and E134 isolates adhered to all surfaces. Various sanitation agents could control biofilms of Geobacillus isolates. The best results were provided by nisin for D413 (80%) and α-amylase for E134 (98%). This paper suggests that sanitation agents could be a solution to control biofilm structures of thermophilic bacilli.