Relative bioefficacy of RRR-α-tocopherol versus all-rac-α-tocopherol in in vitro models

The aim of this study was to evaluate the in vitro relative bioefficacy of RRR-α-tocopherol (RRR- α-T) versus all-rac-α-tocopherol (all-rac-α-T) in counteracting the cytotoxic effect induced by H2O2 in Bovine Mammary Epithelium – University of Vermont (BME-UV1) and Madin-Darby Canine Kidney (MDCK) cells. The range of RRR- α-T and all-rac- α-T concentrations selected for the oxidative challenge experiments was 100µM - 1nM. To study the bioefficacy of RRR- α-T and all-rac- α-T, MTT and LDH tests were performed. Cells were pre-incubated for 3 h with  selected a-tocopherol concentrations and then exposed to increasing H2O2 concentrations ranging from 125 to 750µM for the following 24h. Concerning the cell viability, the pre-treatments with 100µM of RRR- α-T and 100µM all-rac-α-T were able to significantly (P<0.05) counteract the effect induced by 750 µM of H2O2 in BME-UV1. In MDCK the pre-treatment with 1nM of all-rac-α-T was able to significantly (P<0.05) reduce the effect of 125 and 150 mM H2O2. In MDCK cells, the pre-incubation with all-rac-α-T determines a significant reduction of the membrane damage, induced by 175 µM of H2O2. In conclusion, RRR-α-T and all-rac-α-T have shown the ability to counteract the oxidative effects of H2O2, however further investigation will help to better understand their specific mechanism of action in vitro.  

Effect of Zinc Oxide and Zinc Chloride on Human and Swine Intestinal Epithelial Cell Lines

Zinc (Zn) salts are often used as nutritional additives in order to promote gut health. The aim of the present study was to assess the effect of two widely used additives in feedstuff, on the intestinal epithelium. In particular, the effect of zinc oxide (ZnO) and zinc chloride (ZnCl2) was investigated in human (INT-407) and porcine (IPI-2I) cell line models. The effect of Zn sources on IPI-21 and INT-407 cell lines was evaluated by a colorimetric viability test using an incubation period of 3 and 24 hours under serum-free conditions. INT407 and IPI-2I showed to be a suitable model of the intestine and a simple tool to investigate the role of Zn supplements. INT407 showed to be the most sensible model to Zn supplements considered, whereas IPI-2I were more resistant. The results of this study contribute to determine the role of zinc in human and swine intestinal epithelium. However, further in vivo experiments may be done to clarify the contribution of Zn supplements in gut health and to improve Zn supplementation in animal feed and in human formulations

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

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

Rumen-protected choline and vitamin E supplementation in periparturient dairy goats: effects on milk production and folate, vitamin B12 and vitamin E status

We investigated the effects of rumen-protected choline (RPC) and vitamin E (VITE) administration on milk production and status of folate, vitamin B12 and vitamin E during the periparturient period of dairy goats. Forty-eight Saanen multiparous goats were selected for the 72-day experiment, being moved to a maternity pen 30 days before expected parturition and assigned to one of the four experimental groups: control (CTR), no choline or vitamin E supplementation; choline (RPC), supplemented with 4 g/day choline chloride in rumen-protected form; vitamin E (VITE), supplemented with 200 IU/day vitamin E in rumen-protected form; and choline and vitamin E (RPCE), supplemented with 4 g/day RPC chloride and 200 IU/day vitamin E. Supplements were administered individually before the morning feed to ensure complete consumption, starting 30 days before kidding and continuing for 35 days after. During the experiment, milk yield and 4% fat-corrected milk (FCM) yield were, respectively, 210 and 350 g/day higher in RPC-supplemented goats than in non-supplemented goats. Milk fat concentration and fat yield were also increased by RPC treatment. Milk yield and composition were unaffected by vitamin E supplementation. There were no significant interactions between RPC and VITE for any of the variables measured. Plasma metabolites did not differ between treatments before and after kidding except that plasma folate at parturition was higher in RPC-supplemented goats. Neither choline nor vitamin E affected vitamin B12 plasma concentrations, while a time effect was evident after the second week of lactation, when B12 levels in each treatment group started to increase. Vitamin E administration resulted in plasma α-tocopherol levels that were 2 to 2.5 times higher than in non-supplemented goats. Overall, these results suggest that greater choline availability can improve milk production and methyl group metabolism in transition dairy goats

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

Alpha-Tocopherol Counteracts the Cytotoxicity Induced by Ochratoxin A in Primary Porcine Fibroblasts

The aims of the current study were to determine the half-lethal concentration of ochratoxin A (OTA) as well as the levels of lactate dehydrogenase release and DNA fragmentation induced by OTA in primary porcine fibroblasts, and to examine the role of α-tocopherol in counteracting its toxicity. Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P < 0.01) reduced OTA cytotoxicity, lactate dehydrogenase release and DNA damage in both fibroblast cultures. These findings indicate that α-tocopherol supplementation may counteract short-term OTA toxicity, supporting its defensive role in the cell membrane

In vitro digested milk proteins: Evaluation of angiotensin-1-converting enzyme inhibitory and antioxidant activities, peptidomic profile, and mucin gene expression in HT29-MTX cells

Over the past decades, several studies investigated the health-promoting functions of milk peptides. However, to date many hurdles still exist regarding the widespread use of milk-derived bioactive peptides, as they may be degraded during gastrointestinal digestion. Thus, the aim of our study was to in vitro digest intact whey protein isolate (WPI) and casein proteins (CNP), mimicking in vivo digestion, to investigate their bioactive effects and to identify the potential peptides involved. Whey protein isolate and CNP were digested using a pepsin-pancreatin protocol and ultra-filtered (3-kDa cutoff membrane). A permeate (3 kDa) were obtained. Soy protein was included as a control (CTR). Angiotensin-1-converting enzyme inhibitory (ACE1-I) and antioxidant activity (AOX) were assessed and compared with those observed in undigested proteins and CTR. Furthermore, the permeate was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry (LC-nano ESI MS/MS) using a shotgun peptidomic approach, and retentate was further digested with trypsin and analyzed by MS using a shotgun proteomic approach to identify potentially bioactive peptides. Further, the effects of WPI, CNP, and CTR retentate on cell metabolic activity and on mucus production (MUC5AC and MUC2 gene expression) were assessed in intestinal goblet HT29-MTX-E12 cells. Results showed that WPI permeate induced a significant ACE1-I inhibitory effect [49.2 ± 0.64% (SEM)] compared with undigested WPI, CNP permeate, and retentate or CTR permeate (10.40 ± 1.07%). A significant increase in AOX (1.58 ± 0.04 and 1.61 ± 0.02 µmol of trolox AOX equivalents per mg of protein, respectively) upon digestion was found in WPI. Potentially bioactive peptides associated with ACE1-I and antihypertensive effects were identified in WPI permeate and CNP retentate. At specific concentrations, WPI, CNP, and CTR retentate were able to stimulate metabolic activity in HT29-MTX-E12 cells. Expression of MUC5AC was increased by CNP retentate and unaltered by WPI retentate; MUC2 expression was significantly increased by 0.33 mg/g of CNP and reduced by 1.33 mg/g of CNP. Our results confirm that milk proteins may be rich sources of bioactive compounds, with the greatest beneficial potential of CNP at the intestinal goblet cell level

Biotechnological and Technical Challenges Related to Cultured Meat Production

The constant growth of the population has pushed researchers to find novel protein sources. A possible solution to this problem has been found in cellular agriculture, specifically in the production of cultured meat. In the following review, the key steps for the production of in vitro meat are identified, as well as the most important challenges. The main biological and technical approaches are taken into account and discussed, such as the choice of animal, animal-free alternatives to fetal bovine serum (FBS), cell biomaterial interactions, and the implementation of scalable and sustainable biofabrication and culturing systems. In the light of the findings, as promising as cultured meat production is, most of the discussed challenges are in an initial stage. Hence, research must overcome these challenges to ensure efficient large-scale production

Polyphenolic content and antioxidant activity in agri-food wastes and by-products using different extraction methods

Agri-food wastes (AFW) and by-products chain still have the potential to be reprocessed into other production systems. AFW and by-products may contain components that could be valorised for their bioactivity, such as polyphenols and antioxidant molecules that can be used as a source of functional ingredients for feed industries. However the bioaccessibility of these products are higly variable and dependent on a range of factors, one of the most important being food matrix characteristic.The aim of this study was to determine the total phenolic content and the antioxidant capacity of several AFW (fruit and vegetable waste (FVW), citrus pulp, strawberry and orange dried) and by-products (grape marc, Camilina sativa cake, olive pomace and whey) using different extraction protocols. A total of 24 samples were processed using two different extraction methods: chemical extraction and in vitro physiological extraction. Afterwards, the polyphenolic content was assessed by Folin–Ciocalteu assay while antioxidant capacity was determined by 2, 2-Azino-bis-3 ethylbenzothiazoline-6-sulfonic Acid (ABTS) assay. Soy and wheat samples were included as controls in all the experiments.Results obtained showed that the chemical extracts of by-products and AFW contain different amount of polyphenols; in particular, as expected, the grape marc showed the highest polyphenolic content with a value of 4.5% w/w, followed by Camilina sativa cake, olive pomace, FVW, orange and strawberry dried showed a polyphenolic content of 1.3, 0.7, 1.3, 1.6 and 1.3 %w/w, respectively. Considering the antioxidant capacity, grape marc exhibited a significant (P&lt;0.05) value of 573.6 μmol Trolox equivalent/g after chemical extraction compared to the other samples considered. The physiological extraction yielded high polyphenolic content and antioxidant capacity, suggesting that during the digestion the bioaccessibility of phenolic and antioxidant compounds was improved. The results obtained in this study indicate that AFW and by-products could be considered a promising source of antioxidants and phenolic compounds