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Objectives. Develop a Cell Surface Display system in S. cerevisiae, based on the construction of an expression cassette for pYES2 plasmid.Results. The construction of an expression cassette containing the α-factor signal peptide and the Cterminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin, allowing cell surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was con rmed by dot blot, and indirect immuno uorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD.Conclusions. These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

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Aim The aim of the present study was to examine the vaccine immune response in ewes supplemented with Bacillus toyonensis BCT‐7112T during a period of 5‐day supplementation before vaccination against a recombinant Clostridium perfringens epsilon toxin (rETX). Methods and Results Ewes were vaccinated with 200 µg of rETX adjuvanted with 10% aluminium hydroxide. The treat group was orally supplemented with B. toyonensis BCT‐7112T (3 × 108 viable spores) for 5 days prior to the first and second vaccination. Ewes supplemented with B. toyonensis BCT‐7112T showed higher neutralizing antibody titres than the non‐supplemented ewes (P < 0·05), with an increase in serum levels for total IgG anti‐rETX by 3·2‐fold (P < 0·0001), and for both IgG isotypes IgG1 and IgG2 by 2·1‐fold and 2·3‐fold (P < 0·01), respectively, compared with the control group. The peripheral blood mononuclear cells of ewes in the supplemented group had a higher (P < 0·05) cytokine mRNA transcription levels for IL‐2 (6·4‐fold increase), IFN‐γ (2·9‐fold increase) and transcription factor Bcl6 (2·3‐fold increase) compared with the control group. Conclusion We conclude that a 5 days of supplementation with B. toyonensis BCT‐7112T prior vaccination is sufficient to significantly improve the humoral immune response of ewes against C. perfringens recombinant ETX vaccine. Significance and Impact of the study These findings open a new perspective in the utilization of B. toyonensis BCT‐7112T as an immunomodulator since a 5 days period of probiotic supplementation is sufficient to improve the vaccine immune response.

Non-conventional yeasts can be isolated from a wide range of environmental sources, often found in beverage industry in mixed fermentations, in which the microorganisms’ inoculum usually is not fully known. It is important to know starter cultures, since in addition to favoring reproducibility, other properties can be discovered. Thus, the objective of this work was to identify and characterize yeasts isolated from environment, evaluating their probiotic potential and possible use in brewery. Isolates were obtained from flowers, fruits, leaves and mixed-fermentation beers, being identified by PCR. Yeasts with promising activity were evaluated regarding their growth under different pHs, temperature and presence of organic acids. To explore probiotic potential, in vitro tests were performed of antimicrobial activity and co-aggregation with food pathogens, auto-aggregation, and survival in simulated gastrointestinal tract conditions. In our study, Pichia kluyveri (LAR001), Hanseniaspora uvarum (PIT001) and Candida intermedia (ORQ001) were selected among 20 isolates. P. kluyveri was the only one that tolerated pH 2.5. Lactic acid was not inhibitory, while acetic acid and incubation at 37 °C had a partially inhibitory effect on yeasts growth. All yeasts tolerated α-acids from hops and NaCl up to 1%. It is suggested that isolates are able to adhere to intestinal cells and influence positively the organism in combating pathogens, as they showed auto-aggregation rates above 99% and antagonistic activity to pathogenic bacteria. The yeasts tolerated gastric environment conditions, however were more sensitive to pancreatic conditions. We conclude that isolated non-conventional yeasts showed probiotic potential and promising application in beer fermentation.

Non-conventional yeasts can be isolated from a wide range of environmental sources, often found in beverage industry in mixed fermentations, in which the microorganisms' inoculum usually is not fully known. It is important to know starter cultures, since in addition to favoring reproducibility, other properties can be discovered. Thus, the objective of this work was to identify and characterize yeasts isolated from environment, evaluating their probiotic potential and possible use in brewery. Isolates were obtained from owers, fruits, leaves and mixed-fermentation beers, being identi ed by PCR. Yeasts with promising activity were evaluated regarding their growth under different pHs, temperature and presence of organic acids. To explore probiotic potential, in vitro tests were performed of antimicrobial activity and co-aggregation with food pathogens, auto-aggregation, and survival in simulated gastrointestinal tract conditions. In our study, Pichia kluyveri (LAR001), Hanseniaspora uvarum (PIT001) and Candida intermedia (ORQ001) were selected among 20 isolates. P. kluyveri was the only one that tolerated pH 2.5. Lactic acid was not inhibitory, while acetic acid and incubation at 37 °C had a partially inhibitory effect on yeasts growth. All yeasts tolerated α-acids from hops and NaCl up to 1%. It is suggested that isolates are able to adhere to intestinal cells and in uence positively the organism in combating pathogens, as they showed autoaggregation rates above 99% and antagonistic activity to pathogenic bacteria. The yeasts tolerated gastric environment conditions, however were more sensitive to pancreatic conditions. We conclude that isolated non-conventional yeasts showed probiotic potential and promising application in beer fermentation.