Characterization of kefir yeasts with antifungal capacity against Aspergillus species.

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them-belonging to S. cerevisiae and P. kudriavzevii species-generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.

The synthesis of soluble and volatile bioactive compounds by selected brewer's yeasts: Antagonistic effect against enteropathogenic bacteria and food spoiler - toxigenic Aspergillus sp.

Contamination by Aspergillus sp. and the accumulation of its mycotoxins in food and beverages have a high impact on human health and food safety. This investigation inquires the ability of brewer's yeasts discarded after fermentation (brewing fermentation residue, BFR) to synthesize bioactive compounds and to biocontrol Aspergillus sp. BFRs of Saccharomyces cerevisiae MBELGA62 and Pichia kudriavzevii MBELGA61 proved to have bacteriostatic properties and to be efficient in fungal growth reduction, decreasing the growth rate of Aspergillus flavus and Aspergillus parasiticus up to 37.8% and 42.5%, respectively. Fungal mycelium degradation along with absentia of conidia was detected near the yeast inoculum. Moreover, the yeasts synthesize volatile bioactive compounds that extend Aspergillus sp. lag phase above 100% and decrease fungal growth rates from 20% towards 44%, along with the complete inhibition of conidia synthesis. These results indicate the potential of this residue to be used in biocontrol applications in the food industry.

Antifungal effect of kefir fermented milk and shelf life improvement of corn arepas.

Fungal contamination negatively affects the production of cereal foods such as arepa loaf, an ancient corn bread consumed daily in several countries of Latin-America. Chemical preservatives such as potassium sorbate are applied in order to improve the arepa's shelf life and to reduce the health risks. The use of natural preservatives such as natural fermented products in food commodities is a common demand among the consumers. Kefir is a milk fermented beverage obtained by fermentation of kefir grains. Its antibacterial and probiotic activity has been exhaustively demonstrated. Our objectives were to determine the antifungal effect of kefir fermented milk on Aspergillus flavus AFUNL5 in vitro and to study if the addition of kefir fermented milk to arepas could produce shelf life improvement. We determined the antifungal effect on solid medium of kefir cell-free supernatants (CFS) obtained under different fermentation conditions. Additionally, we compared the antifungal effect of kefir CFS with that obtained with unfermented milk artificially acidified with lactic plus acetic acids (lactic and acetic acids at the same concentration determined in kefir CFS) or with hydrochloric acid. Finally, kefir was added to the corn products either in the loaf recipe (kefir-baked arepas) or sprayed onto the baked-loaf surface (kefir-sprayed arepas). The loaves' resistance to natural and artificial fungal contamination and their organoleptic profiles were studied. The highest fungal inhibition on solid medium was achieved with kefir CFS produced by kefir grains CIDCA AGK1 at 100 g/L, incubated at 30 °C and fermented until pH 3.3. Other CFS obtained from different fermentation conditions achieved less antifungal activity than that mentioned above. However, CFS of milk fermented with kefir grains, until pH 4.5 caused an increase of growth rates. Additionally, CFS produced by kefir grains CIDCA AGK1 at 100 g/L, incubated at 30 °C and fermented until pH 3.3 achieved higher antifungal activity than CFS from artificially acidified milk with organic acids (CFS L + A) at the same concentration of kefir CFS. Besides, CFS from milk acidified with hydrochloric acid (CFS HCl) showed no fungal inhibition. On the other hand, kefir-baked arepas exhibited significant resistance to natural and artificial fungal contamination. Finally, both kefir-baked and kefir-sprayed arepas retained the organoleptic characteristics of the traditional corn product, but with certain tastes imparted by the kefir fermentation. This work constitutes the first study on fungal inhibition by kefir-fermented milk extending to the protection of corn products of mass-consumption and the possible application as a food preservative.

Characterization of kefir yeasts with antifungal capacity against Aspergillus species / Caracterización de levaduras de kéfir con capacidad antifúngica frente a especies de Aspergillus

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them—belonging to S. cerevisiae and P. kudriavzevii species—generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.(AU)

Inhibición del crecimiento de Aspergillus ochraceus mediante "panela" fermentada con gránulos de kefir de agua / Fungal growth inhibition of Aspergillus ochraceus with "panela" fermented with water kefir grains

Antecedentes: El kéfir de agua (KA) es una bebida producida con soluciones azucaradas fermentadas con gránulos constituidos por bacterias ácido lácticas (BAL) y levaduras embebidas en una matriz de polisacárido. Aspergillus ochraceus es un hongo filamentoso micotoxigénico, contaminante frecuente y productor de ocratoxina en café, uvas y vino. Se ha estudiado la actividad antifúngica de productos fermentados por BAL pero hasta ahora no se conocen estudios sobre la capacidad antifúngica del KA. Objetivos: Evaluar la capacidad antifúngica de soluciones de panela fermentadas a diferentes tiempos y temperaturas con KA sobre A. ochraceus. Métodos: Se inocularon 10 µL de una suspensión (3x107 conidios. ml-1) de A. ochraceus en agar malta mezclado con sobrenadantes libres de células (SLC) de los productos fermentados, incubando a 25°C y midiendo diariamente el diámetro del micelio, con lo cual se determinó el efecto antifúngico sobre la tasa de crecimiento y la fase de latencia. Se determinó la concentración de ácidos orgánicos (AO) en los SLC mediante HPLC. Resultados: Los productos fermentados por mayor tiempo y temperatura tuvieron mayor concentración de (AO) y mayor descenso de pH siendo el SLC8 (pH: 2,8) el más inhibitorio. Conclusiones: La solución de panela en agua fue adecuada para fermentación con gránulos de KA, obteniéndose metabolitos con propiedades antifúngicas como los AO.