Selección de cepas nativas de Lactobacillus con actividad inhibitoria y tolerantes al etanol aisladas de "masato"

Abstract

La conservación de alimentos por fermentación natural es ampliamente practicada por diversas comunidades nativas. El “masato” es producto de este proceso y constituye parte importante de la alimentación de los pobladores de la amazonía peruana. La masa de yuca, mandioca o cassava (Manihot esculenta, Crantz) utilizada en la preparación de esta bebida tradicional, constituye el substrato principal para el establecimiento de una microflora mixta ambiental, incluyendo las bacterias lácticas como responsables de la fermentación maloláctica, láctica y otras propiedades de interés industrial. La búsqueda de nuevas cepas nativas de bacterias lácticas productoras de sustancias antimicrobianas, adaptadas a las condiciones ambientales y materias primas regionales, es sumamente importante, para mejorar la calidad sensorial y sanitaria del “masato”. Para detectar cepas lácticas con propiedades antimicrobianas, se procesaron 11 muestras de “masato” procedentes de Pucallpa – Ucayali, las cuales fueron sembradas en agar Man Rogosa Sharpe (MRS) pH 6.5 e incubadas a 30° C por 72 horas en condiciones de microaerofilia. La caracterización morfológica, cultural, bioquímica y la actividad antimicrobiana se realizaron según Kandler Weiss (1986) y Tagg McGiven, (1971) respectivamente. De un total de 81 cepas de Lactobacillus aisladas, se identificaron 33 (41%) de Lactobacillus plantarum, 13 (16%) de Lactobacillus alimentarius, 12 (15%) de Lactobacillus acidophilus, 9 (11%) de Lactobacillus casei y en menores porcentajes Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus amylophilus y Lactobacillus coryniformis. Se encontró además, 64 (78.9%) de Lactobacillus heterofermentativos y 17 (21.1%) de Lactobacillus homofermentativos. En todas las muestras de “masato” evaluadas, predominó Lactobacillus plantarum, seguido de Lactobacillus acidophilus y Lactobacillus alimentarius. De 8 cepas lácticas productoras evaluadas, Lactobacillus plantarum M4 fue la única cepa productora de una sustancia antimicrobiana que no es ácido orgánico ni agua oxigenada. Se demostró además, que la sustancia antimicrobiana neutralizada a pH 6,5 posee un reducido espectro antimicrobiano y es estable en un amplio rango de temperatura y pH. Asimismo, las cepas de Lactobacillus muestran un alto nivel de tolerancia a concentraciones de etanol agregadas al medio de cultivo, siendo mas del 80% de cepas aisladas tolerantes a 10% de etanol, mientras que 6% tolera concentraciones de hasta 12% v/v de etanol. Se proponen las cepas Lactobacillus plantarum 4M0.2, Lactobacillus plantarum M9.5, Lactobacillus acidophilus M2.9, Lactobacillus alimentarius 5M1.2 como una selección de cepas de interés biotecnológico, así como a Lactobacillus plantarum M4, cepa del estudio con mayor espectro antimicrobiano tolerante hasta una concentración de 8% v/v de etanol en el medio de cultivo.

The food preservation by natural fermentation is practiced by diverse native communities. The "masato" is product of this process and constitutes important part of the feeding of the population of peruvian amazonia. The mass of yucca, mandioca or cassava (Manihot esculenta, Crantz) used in the preparation of this traditional drink, constitutes the main substrate for the establishment of an environmental mixed microflora, including the lactic acid bacteria which are responsible for the malolactic and lactic fermentation and other properties of industrial interest. The search of new native strains of antimicrobial substances producing lactic bacteria, adapted to the environmental conditions and regional raw materials, is extremely important, to improve the sensorial and sanitary quality of the "masato". In order to detect lactic strains with antimicrobial properties, 11 samples of "masato" coming from Pucallpa - Ucayali were processed, which were seeded in agar Man Rogosa Sharpe (MRS) pH 6,5 and incubated to 30° C by 72 hours in microaerophilic conditions. The morphologic, cultural, biochemical characterization and the antimicrobial activity were made according to Kandler Weiss (1986) and Tagg McGiven, (1971) respectively. Of a total of 81 isolated strains of Lactobacillus, 33 (41%) of Lactobacillus were identified like Lactobacillus plantarum, 13 (16%) like Lactobacillus alimentarius, 12 (15%) like Lactobacillus acidophilus, 9 (11%) like Lactobacillus casei and in smaller percentage like Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus amylophilus and Lactobacillus coryniformis. In addition, 64 (78.9%) and 17 (21.1%) of Lactobacillus were detected homofermenters and heterofermenters respectively. In all the evaluated samples of "masato", predominated Lactobacillus plantarum, followed of Lactobacillus acidophilus and Lactobacillus alimentarius. Of 8 evaluated antimicrobial substance producing lactic acid bacteria strains, Lactobacillus plantarum M4 was the only producing strain of a antimicrobial substance that is not acid organic nor oxygenated water. In addition, it was shown that the neutralized antimicrobial substance to pH 6.5 has a reduced antimicrobial spectrum and is stable in a wide range of temperature and pH. Also, the strains of Lactobacillus showed a high level of tolerance to concentrations of ethanol added to culture means, being but of 80% of tolerant isolated strains to 10% of ethanol, whereas 6% tolerated concentrations of up to 12% v/v of ethanol. We propose to Lactobacillus plantarum 4M0.2, Lactobacillus plantarum M9.5, Lactobacillus acidophilus M2.9, Lactobacillus alimentarius 5M1.2 like a selection of strains of biotechnological interest, as well as to Lactobacillus plantarum M4, strain of the study with greater antimicrobial spectrum and tolerance until 8% v/v concentration of ethanol added in culture mean.