Lactobacillus pentosus ABHEAU-05: An in vitro digestión resistant lactic acid bacterium isolated from a traditional fermented Mexican beverage / Lactobacillus pentosus ABHEAU-05: una bacteria ácido láctica resistente a la digestión in vitro aislada de una bebida fermentada tradicional mexicana

Abstract The health benefits attributed to probiotics generate interest in the search of competent strains adapted to several ecological niches, especially those related to traditional beverages and foods of each country. Pineapple tepache, a traditional Mexican fermented beverage, was used for the isolation of lactic acid bacteria with probiotic potential, one of which withstood the in vitro tests. The isolated strain AB-05, which exhibited the tested probiotic functional properties, was designated as Lactobacillus pentosus ABHEAU-05. The sequence was registered in GenBank under access code MK587617. This study is the first report of a lactic acid bacterium with in vitro digestion resistance isolated from pineapple tepache. The survival of L. pentosus ABHEAU-05 in a symbiotic medium was proven using fermented milk enriched with inulin. The in vitro digestion-resistant probiotic activity of lactobacilli was measured through analysis of pH and proteolysis. Results showed that L. pentosus grew properly in fermented milk; therefore, this microorganism could be used in the manufacture of this kind of products. The concentration of L. pentosus reached up to 8.5 logCFU/ml after 40 h of fermentation. In addition, the production of peptides and the decrease in pH indicated the vigorous and active metabolic state of the lactic acid bacterium tested. The activity and the concentration of this microorganism were maintained during refrigeration. The results of this research conclude that L. plantarum ABHEAU-05 is an in vitro digestion-resistant microorganism that can be used as a starter culture for the production of functional foods of dairy origin.

Resumen Los beneficios a la salud atribuidos a los probióticos generan interés en la búsqueda de cepas competentes adaptadas a varios nichos ecológicos, especialmente los relacionados con bebidas y alimentos tradicionales de cada país. En este estudio, se aisló del tepache de pina, una bebida fermentada tradicional mexicana, una bacteria láctica resistente a la digestión in vitro. Entre 5 bacterias aisladas, una de ellas soportó las pruebas simuladas de digestión gastrointestinal. Se analizó la resistencia a sales biliares, a condiciones ácidas y al ataque enz-imático con pepsina. La bacteria aislada, que exhibió las propiedades funcionales probióticas referidas, fue identificada como Lactobacillus pentosus y designada como L. pentosus ABHEAU-05. La secuencia fue depositada en GenBank (acceso MK587617). Se comprobó la supervivencia de L. pentosus ABHEAU-05 en una leche fermentada adicionada con inulina y su resistencia a la digestión in vitro mediante el análisis del pH y la proteólisis. Los resultados muestran que la leche fermentada es una matriz adecuada, donde L. pentosus ABHEAU-05 se desarrolla sin inconvenientes, alcanzando un título de 8,5 logufc/ml después de 40 h de fermentación. Además, la producción de péptidos y el descenso del pH indicaron el estado metabólico vigoroso y activo del microorganismo probiótico. Se concluye que L. pentosus ABHEAU-05 es un microorganismo resistente a la digestión in vitro, que puede servir como cultivo iniciador para la producción de alimentos de origen lácteo. Este es el primer informe acerca del aislamiento de una bacteria ácido láctica resistente a la digestión in vitro a partir del tepache de piña.

Evaluación de las propiedades bioactivas in vitro de bacterias ácido lácticas aisladas de nichos ecológicos nativos del Ecuador / Assessment of the in vitro bioactive properties of lactic acid bacteria isolated from native ecological niches of Ecuador

Lactic acid bacteria are known for their biotechnological potential. In various regions of Ecuador numerous indigenous biological resources are largely undocumented. In this study, we evaluated the potential probiotic characteristics and antagonistic in vitro properties of some lactic acid bacteria from native niches of the subtropical rain forests of Ecuador. These isolates were identified according to their morphological properties, standard API50CH fermentation profile and RAPD-DNA polymorphism pattern. The selected isolates were further evaluated for their probiotic potential. The isolates grew at 15 °C and 45 °C, survived at a pH ranging from 2.5 to 4.5 in the presence of 0.3% bile (>90%) and grew under sodium chloride conditions. All selected isolates were sensitive to ampicillin, amoxicillin and cefuroxime and some showed resistance to gentamicin, kanamycin and tetracycline. Moreover, the agar well diffusion assay showed that the supernatant of each strain at pH 3.0 and pH 4.0, but not at pH 7.0 exhibited increased antimicrobial activity (inhibition zone >15 mm) against two foodborne pathogens, Escherichia coli and Salmonella spp. To our knowledge, this is the first report describing the antagonistic activity against two foodborne pathogens and the probiotic in vitro potential of lactic acid bacteria isolated from native biota of Ecuador.

Las bacterias ácido lácticas (BAL) son conocidas por su potencial biotecnológico. En diversas regiones del Ecuador existen recursos biológicos nativos, que en su mayoría no han sido documentados. En este estudio se evaluaron in vitro las posibles características probióticas y antagónicas de algunas BAL aisladas de nichos nativos de la selva subtropical. Estas cepas se clasificaron en función de sus propiedades morfológicas, el perfil estándar de fermentación API50CH y los patrones de polimorfismo RAPD-ADN. Diez microorganismos fueron seleccionados y se probó su potencial probiótico. Todas las bacterias crecieron a 15 °C y 45 °C, sobrevivieron en el rango de pH ácido de 2,5 a 4,5 en presencia de 0,3% de bilis (> 90%), mostraron tolerancia a los tratamientos en cloruro de sodio y diferentes perfiles de sensibilidad a antibióticos. Todas las cepas fueron sensibles a ampicilina, amoxicilina y cefuroxima, y algunas fueron resistentes a gentamicina, kanamicina y tetraciclina. Por otra parte, el ensayo de pruebas de difusión en agar mostró que el sobrenadande de cada cepa cultivada a pH 3,0 y pH 4,0, pero no a pH 7,0, presentó elevada actividad antimicrobiana (zona de inhibición > 15 mm) frente a 2 agentes patógenos alimentarios, Escherichia coli y Salmonella spp. Este estudio describe por primera vez la actividad antagonista frente a patógenos de origen alimentario y el potencial probiótico in vitro de BAL aisladas de nichos ecológicos nativos del Ecuador.

Propriedades probióticas in vitro de Lactobacillus spp. isolados de queijos minas artesanais da Serra da Canastra - MG / In vitro probiotic properties of Lactobacillus spp. isolated from Minas artisanal cheese from Serra da Canastra - MG

O objetivo deste estudo foi determinar o potencial probiótico in vitro de Lactobacillus spp. isolados de queijos minas artesanais da Serra da Canastra, considerando-se o antagonismo entre amostras isoladas frente a microrganismos indicadores, a susceptibilidade a antimicrobianos, a sensibilidade ao ácido gástrico e a sensibilidade a sais biliares. Todas as bactérias ácido-lácticas testadas apresentaram resistência ao ácido gástrico (pH 2,0) e aos sais biliares (0,3%), bem como atividade antagonista contra Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica var. Typhimurium, Enterococcus faecalis e bactérias ácido-lácticas isoladas dos próprios queijos - Lactobacillus plantarum (D27) e Lactobacillus rhamnosus (B25). Todas as amostras foram sensíveis à eritromicina e tetraciclina e resistentes à ciprofloxacina, gentamicina, oxacilina, estreptomicina e vancomicina. L. plantarum (B17) apresentou melhor potencial probiótico, pois obteve resultados satisfatórios em todas as propriedades avaliadas. Mais estudos são necessários para verificar a presença e a capacidade de transmissão de genes de resistência antimicrobiana a outros microrganismos e para avaliar o potencial dos microrganismos in vivo. As bactérias selecionadas poderão ser utilizadas na elaboração de queijos em que sejam mantidos o sabor e a tradição do queijo minas artesanal do estado de Minas Gerais...

The aim of this study was to determine some in vitro probiotic features of Lactobacillus spp. isolated from Minas artisanal cheese from Serra da Canastra in order to select some bacteria for future production of safer cheeses keeping their natural flavor and tradition. The evaluated properties were antagonism against indicator microorganisms, antimicrobial susceptibility and also sensitivity to gastric acid and to bile salts. All lactic acid bacteria tested were resistant to gastric acid (pH 2.0) and bile salts (0.3%). Antagonistic activities were detected against Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella entericavar. Typhimurium, Enterococcus faecalis and other lactic bacteria isolated from the same cheese - Lactobacillus plantarum (D27) and Lactobacillus rhamnosus (B25). All samples were sensitive to the antimicrobials erythromycin, tetracycline and resistant to ciprofloxacin, gentamycin, oxacillin, streptomycin and vancomycin. L.plantarum (B17) presented the best probiotic potential. Further studies are needed to verify the presence and the capacity of transmission of antimicrobial resistance genes to other microorganisms and evaluate the in vivo probiotic potential of the selected microorganisms...

Isolation, enumeration, molecular identification and probiotic potential evaluation of lactic acid bacteria isolated from sheep milk

Lactic acid bacteria species were molecularly identified in milk from Lacaune, Santa Inês and crossbred sheep breeds and their in vitro probiotic potential was evaluated. The species identified were Enterococcus faecium (56.25%), E. durans (31.25%) and E. casseliflavus (12.5%). No other lactic acid bacteria species, such as lactobacilli, was identified. Most of the isolated enterococci were resistant to gastric pH (2.0) and to 0.3% oxgall. All tested enterococci were resistant to ceftazidime, oxacillin and streptomycin and sensible to clindamycin, erythromycin and penicillin. The resistance to ciprofloxacin, gentamicin, tetracycline and vancomycin varied among tested species. All tested enterococci strongly inhibited (P<0.05) Escherichia coli and Listeria monocytogenes, moderately inhibited E. faecalis and Staphylococcus aureus and did not inhibit Pseudomonas aeruginosa, Salmonella enterica var. Typhimurium and also one E. durans sample isolated from sheep milk. Four samples of E. faecium, one of E. durans and one of E. casseliflavus presented the best probiotic potential...

Espécies de bactérias ácido-lácticas foram identificadas em nível molecular em leite das raças ovinas Lacaune, Santa Inês e suas mestiças, e o seu potencial probiótico in vitro foi avaliado. As espécies identificadas foram Enterococcus faecium (56,25%), E. durans (31,25%) e E. casseliflavus (12,5%). Nenhuma outra espécie de bactéria ácido-láctica, como Lactobacillus sp., foi identificada. A maioria dos enterococos isolados foi resistente ao pH gástrico (2.0) e a 0,3% de oxgall. Todos os enterococos testados foram resistentes à ceftazidima, oxacilina e estreptomicina e sensíveis à clindamicina, eritromicina e penicilina. A resistência à ciprofloxacina, gentamicina, tetraciclina e vancomicina variou entre as amostras. Todos os enterococos testados inibiram fortemente (P<0,05) Escherichia coli e Listeria monocytogenes, inibiram moderadamente E. faecalis e Staphylococcus aureus e não inibiram Pseudomonas aeruginosa, Salmonella enterica var. Typhimurium e uma amostra de E. durans isolada de leite de ovelha. Quatro amostras de E. faecium, uma de E. durans e uma de E. casseliflavus apresentaram o melhor potencial probiótico...

Potencial probiótico in vitro de bactérias ácido-láticas isoladas de queijo-de-minas artesanal da Serra da Canastra, MG / Probiotic potential of lactic acid bacteria isolated from minas artisanal cheese from Serra da Canastra, MG

O potencial probiótico in vitro de 12 amostras de bactérias ácido-láticas (11 Lactobacillus spp. e uma Weissella paramesenteroides), isoladas de queijo-de-minas artesanal da Serra da Canastra, foi investigado. Essas amostras foram caracterizadas in vitro quanto à susceptibilidade a antimicrobianos, antagonismo contra microrganismos de referência patogênicos e não patogênicos e sensibilidade a pH gástrico e sais biliares. Apenas L. rhamnosus B4, W. paramesenteroides C10 e L. rhamnosus D1 apresentaram resistência a menor número de antimicrobianos (ceftazidima, oxacilina, estreptomicina e vancomicina). Todas as amostras apresentaram atividade antagonista frente a todas as bactérias patogênicas testadas e não inibiram as bactérias não patogênicas. Todas as amostras foram resistentes ao pH gástrico (2.0). Alguns microrganismos mostraram pouca inibição do crescimento em presença de sais biliares, enquanto outros foram moderadamente ou altamente inibidos. L. rhamnosus B4, W. paramesenteroides C10 e L.rhamnosus D1 apresentaram o melhor potencial probiótico de acordo com os testes in vitro realizados.

The in vitro probiotic potential of twelve samples of lactic acid bacteria (eleven Lactobacillus spp. and one Weissella paramesenteroides) isolated from Minas artisanal cheese from Serra da Canastra was evaluated for their antimicrobial susceptibility, antagonistic activity against pathogenic and lactic acid bacteria, as well as for their tolerance to gastric pH and biliary salts. Only L. rhamnosus B4, W. paramesenteroides C10, and L. rhamnosus D1 showed resistance to tested antimicrobials (ceftazidime, oxacillin, streptomycin, and vancomycin). All samples showed antagonism against all pathogenic bacteria tested and did not inhibit the growth of nonpathogenic samples. Regarding gastric pH tolerance, all samples showed low inhibition at pH 2. The tolerance to biliary salts varied from low to great among tested samples. L. rhamnosus B4, W. paramesenteroides C10, and L. rhamnosus D1 showed the best probiotic potential revealed by their in vitro test results.