In vitro evaluation of antimicrobial and antioxidant activities of algal extracts

The aim of this study was to evaluate the antioxidant capacity, the antimicrobial proprieties of algae Ascophyllum nodosum and Schizochytrium spp. against one of major swine enteric pathogen Escherichia coli O138 by broth macro-dilution method in Luria-Bertani (LB) medium. The antimicrobial effect of the algal extracts at supplementation of 0.12%, 0.06% and 0.03% (v/v) on E. coli O138, genetically characterised by PCR, was evaluated by following the bacterial growth. The antioxidant activity was determined by the ABTS Radical Cation Decolorisation Assay. In particular, the log(10) E. coli used as control resulted significantly higher than 0.12% at 3 hours (8.82 +/- 0.07 and 8.18 +/- 0.07 log(10) cells/mL, respectively; p<.01) suggesting an inhibitory activity related to the dose. No effect activity was observed with Schizochytrium spp. against E. coli growth. A. nodosum and Schizochytrium spp. exhibited antioxidant capacity (p<.05). The combination of them (1:1) exhibited antioxidant activity suggesting a synergistic effect (p<.05). The different proprieties of algal species that can modulate the O138 E. coli growth, one of the major pathogen of swine species, together with the antioxidant capacity, make them a promising functional feed additive to improve the gut health, therefore further studies are needed to confirm these activities in vivo

Retarded germination of Nicotiana tabacum seeds following insertion of exogenous DNA mimics the seed persistent behavior

Tobacco seeds show a coat-imposed dormancy in which the seed envelope tissues (testa and endosperm) impose a physical constraint on the radicle protrusion. The germination-limiting process is represented by the endosperm rupture which is induced by cell-wall weakening. Transgenic tobacco seeds, obtained by insertion of exogenous genes codifying for seed-based oral vaccines (F18 and VT2eB), showed retarded germination with respect to the wild type and modified the expression of endogenous proteins. Morphological and proteomic analyses of wild type and transgenic seeds revealed new insights into factors influencing seed germination. Our data showed that the interference of exogenous DNA influences the germination rather than the dormancy release, by modifying the maturation process. Dry seeds of F18 and VT2eB transgenic lines accumulated a higher amount of reserve and stressâ\u80\u93related proteins with respect to the wild type. Moreover, the storage proteins accumulated in tobacco F18 and VT2eB dry seeds have structural properties that do not enable the early limited proteolysis observed in the wild type. Morphological observations by electron and light microscopy revealed a retarded mobilization of the storage material from protein and lipid bodies in transgenic seeds, thus impairing water imbibition and embryo elongation. In addition, both F18 and VT2eB dry seeds are more rounded than the wild type. Both the morphological and biochemical characteristics of transgenic seeds mimic the seed persistent profile, in which their roundness enables them to be buried in the soil, while the higher content of storage material enables the hypocotyl to elongate more and the cotyledons to emerge

Evaluation of antigens stability of tobacco seeds as edible vaccine against VTEC strains

Plants have represent a promising alternative for biopharmaceutical proteins (Ma et al., 2003; Rossi et al., 2014). Many plant based edible vaccines have been shown to be effective in inducing local immune responses (Rossi et al., 2013). Edible vaccines can activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining. This dual effect would provide first-line defense against pathogens invading through the mucosa. The antigens are released in the intestines are taken up by M cells that are present over the Payer’s patches (in the ileum) and the gut associated lymphoid tissue (GALT). Edible vaccines represent an important worldwide goal for the prevention of the enteric diseases, also in livestock. In particular, the enteric infections are a significant clinical problem in pigs. Verocytotoxic Escherichia (E.) coli strains are responsible for serious enterotoxaemia that causes important economic losses in the pig industry. The production of a vaccine for oral administration of transgenic seeds could be a practical and efficient system to prevent the infection and to reduce the antibiotic use. This study was focused on tobacco plants, previously transformed by agroinfection for the seed-specific expression of antigenic proteins (F18 adhesive fimbriae and the B subunit of the Vt2e toxin) as model of edible vaccines against verocytotoxic E. coli strains. The dietary administration of transgenic tobacco seeds promotes a significant increase in the number of mucosal IgA-producing cells of the tunica propria in both small and large intestine in mice (Rossi et al., 2013). A protective effect of oral administration of transgenic tobacco seeds was also observed against verocytotoxic Escherichia coli infection in piglets (Rossi et al., 2014). The aim of this study was to assess the seed-expression stability, that is a important requirement in the vaccine production, of F 18 and Vt2e-B heterologous genes into the progeny of transformed tobacco plants

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection

Antioxidant and Antimicrobial Activity of Algal and Cyanobacterial Extracts: An In Vitro Study

Algae and cyanobacteria, other than their nutritional value, possess different beneficial properties, including antioxidant and antimicrobial ones. Therefore, they can be considered functional ingredients in animal feed and natural substitutes for antibiotics. The aim of this study was to evaluate the antioxidant and antimicrobial capacity against porcine O138 E. coli of Ascophyllum nodosum, Chlorella vulgaris, Lithotamnium calcareum, Schizochytrium spp. as algal species and Arthrospira platensis as cyanobacteria. The antioxidant capacity was determined by ABTS Radical Cation Decolorization Assay testing at three different concentrations (100%; 75%; 50%). The growth inhibition effect of the extracts at concentrations of 25%, 12.5%, 6%, 3% and 1.5% against porcine O138 E. coli was genetically characterized by PCR to detect the presence of major virulence factors; this was evaluated by following the microdilution bacterial growth method. The ABTS assay disclosed that Ascophyllum nodosum was the compound with the major antioxidant properties (57.75 ± 1.44 percentage of inhibition; p < 0.0001). All the extracts tested showed growth inhibition activity at a concentration of 25%. Among all extracts, A. nodosum was the most effective, showing a significant growth inhibition of E. coli; in particular, the log(10) cells/mL of E. coli used as a control resulted in a significantly higher concentration of 25% and 12.5% after 4 h (8.45 ± 0.036 and 7.22 ± 0.025 log(10) cells/mL, respectively; p < 0.005). This also suggests a dose-dependent relationship between the inhibitory activity and the concentration. Also, a synergistic effect was observed on antioxidant activity for the combination of Ascophyllum nodosum and Lithotamnium calcareum (p < 0.0001). Moreover, to determine if this combination could affect the viability of the IPEC-J2 cells under the normal or stress condition, the viability and membrane integrity were tested, disclosing that the combination mitigated the oxidative stress experimentally induced by increasing the cell viability. In conclusion, the results obtained highlight that the bioactive compounds of algal species are able to exert antioxidant capacity and modulate O138 E. coli growth. Also, the combination of Ascophyllum nodosum and Lithotamnium calcareum species can enhance their bioactivity, making them a promising functional feed additive and a suitable alternative to antibiotics

Lysozyme–Mineral Clay Systems: Comparison of Interaction for Controlled Release in Feed Application

A synthetic approach to bond lysozyme (LY) to commercial natural carriers, namely clay minerals (bentonite, BN; and sepiolite, SP) and commercial zeolite (Phil 75((R)), PH), already in use in feed formulation, is proposed. The synthetic route, which implies solid-liquid adsorption, is a simple and effective way for preparing hybrid materials characterized by LY loadings up to 37 mg(LY)/g(carrier). By operating at pH 4.3, initial LY content of 37.5 mg(LY)/g(carrier), and reaction time of 90 min, hybrid materials with LY loadings of 37, 35, and 12 mg(LY)/g(carrier) for LY-SP, LY-BN, and LY-PH, respectively were obtained. The LY initial concentration and pH, as well as the physico-chemical properties of the carries were found to be the parameters that govern the synthesis of the materials. The driving force for an effective LY adsorption and interaction is the combined Zero Point Charge (ZPC) of the carriers, always negative (in the range between 4 and 170 mV) and the positive ZPC of LY, as well as the carrier morphology, characterized by mesoporosity (pore dimensions in the range of 5-12 nm). However, it is the interaction of charges of opposite sign that mainly affects LY loadings and bond strength. Based on SEM-EDX analysis, LY molecules are quite homogeneously spread onto the carriers' surface. TG-DTG analyses showed that the LY-carrier interaction in the hybrid materials is stronger than that in a simple mechanical mixture of the components. Specifically, in the hybrid materials, the phenomenon at 300 degrees C, associated to LY decomposition, is broadened and slightly shifted towards higher temperatures (320-350 degrees C), whereas in a mechanical mixture of the same composition, it occurs at temperatures closer to those of free LY, as if there were no or very weak interactions. At pH 3, a very little LY release, 0.03 and 0.01 mg(LY)/g(carrier), was found for LY-BN and LY-PH, respectively. The latter became larger at pH 7, 0.06 mg(LY)/g(carrier) for both BN and PH carriers, suggesting that BN and PH are better modulators of LY release. The paper provides insights for the study and the development of new optimized feed formulations for the targeted delivery of natural compounds with antimicrobial activity, alternatives to antibiotics, and vaccinal antigens

Proteomic/peptidomic profile and Escherichia coli growth inhibitory effect of in vitro digested soya protein

Plant proteins contain bioactive peptides with functional properties and physiological activities. In the present work, the bioactive peptides produced during in vitro gastrointestinal digestion of soya protein isolate were investigated. Protein samples were subjected to simulated gastrointestinal digestion with a generation of permeate (3 kDa) fractions. The permeate was analysed by nano-liquid chromatography electrospray ionisation tandem mass spectrometry (LC-nano ESI MS/MS) using a shotgun peptidomic approach, and the retentate was further digested with trypsin and analysed using a shotgun proteomic approach. Based on protein profile observed, the retentate was further tested for its potential antimicrobial activity by evaluating the inhibitory effect on E. coli growth. In the present study the peptidomic/ proteomic characterisation of permeate and retentate fractions revealed the presence of bioactive peptides and proteins associated with antioxidant, ACE-inhibitory, anti-hypertensive and antimicrobial activities. The presence of potentially antimicrobial proteins in the retentate fraction is supported by a marked E. coli F18þ growth inhibitory activity of the same fraction. In particular, the growth inhibitory effect was significant from one until six hours of incubation with 0.65–2.6 mg/ml of in vitro digested soya. The obtained data confirmed that soya-based supplements may have potential beneficial effects after human consumption, and they may be recycled for animal nutrition in line with the circular economy concept