Reevaluating a non-conventional procedure to microencapsulate beneficial lactobacilli: assessments on yield and bacterial viability under simulated technological and physiological conditions.

BACKGROUND: Maintaining viability of beneficial microorganisms applied to foods still constitutes an industrial challenge. Many microencapsulation methodologies have been studied to protect probiotic microorganisms and ensure their resistance from manufacturing through to consumption. However, in many Latin-American countries such as Argentina there are still no marketed food products containing microencapsulated beneficial bacteria. The objectives of this work were: (i) to obtain microcapsules containing Lactobacillus fermentum L23 and L. rhamnosus L60 in a milk protein matrix; and (ii) to evaluate the viability of microencapsulated lactobacilli exposed to long-term refrigerated storage, mid-high temperatures and simulated gastrointestinal conditions. RESULTS: The method of emulsification/rennet-catalyzed gelation of milk proteins used in this study led to high encapsulation yields for both strains (98.2-99%). Microencapsulated lactobacilli remained viable for 120 days at 4 °C, while free lactobacilli gradually lost their viability under the same conditions. Microencapsulation increased the resistance of lactobacilli to mid-high temperatures, since they showed survival rates of 95-99.3% at 50 °C, and of 72.5-74.4% at 65 °C. Under simulated gastric conditions, the microencapsulated lactobacilli counts were higher than 8.5 log CFU mL-1 and showed survival rates between 96.61% and 97.74%. Furthermore, in the presence of bile (0.5-2% w/v) the survival of microencapsulated strains was higher than 96%. CONCLUSION: The microencapsulation process together with the matrix of milk proteins used in this study protected beneficial Lactobacillus strains against these first simulated technological and physiological conditions. These findings suggest that this microencapsulation method could contribute to secure optimal amounts of living lactobacilli cells able to reach the intestine. © 2021 Society of Chemical Industry.

Lactic acid bacteria with promising AFB1 binding properties as an alternative strategy to mitigate contamination on brewers' grains.

Adsorption of molecules to the cell walls of microorganisms plays an important role in helping to prevent animal exposure to the toxic and carcinogenic effects of aflatoxins (AFs). The aim of this study was to evaluate the ability of LAB strains, isolated from brewers' grains, to adsorb aflatoxin B1 (AFB1). All LAB were able to reduce the bioavailability of AFB1 from phosphate buffered-saline (PBS). In addition, the strains retained their effectiveness even after heat treatment. The AFB1-LAB complex stability was first evaluated through sequential washing steps. These assays demonstrated that a low percentage of AFB1 was released after consecutive washes. After subjecting the complex to different pH and bile salt treatments, the percentage of bound AF decreased, as compared to the control, but remained at high levels. Finally, to simulate the formation of the AFB1-LAB complex at conditions similar to those of the gastrointestinal tract, LAB and AFB1 were homogenized in PBS adjusted at acidic conditions or under different bile salt concentrations. In general, LAB strains showed the highest AFB1 adsorption at the lowest pH (2) and bile salt concentration (0.05%). In conclusion, the studied strains represent promising biocontrol agents for preventing and/or ameliorating the AFB1 contamination of feed.

Biological control of AFB1-producing Aspergillus section Flavi strains isolated from brewer's grains, alternative feed intended for swine production in Argentina.

The aim of the present study was to investigate the inhibitory activity of lactic acid bacteria (LAB) isolated from brewer's grains on Aspergillus section Flavi growth and aflatoxin B1 production. The Aspergillus strains tested were inhibited by all the LAB strains assayed. The isolates Lactobacillus brevis B20, P. pentosaceus B86, Lactococcus lactis subsp. lactis B87, L. brevis B131, and Lactobacillus sp. B144 completely suppressed the fungal growth and reduced aflatoxin B1 production. In conclusion, LAB isolated from brewer's grains show a high inhibitory activity on fungal growth and aflatoxin biosynthesis by Aspergillus flavus and Aspergillus parasiticus. Further studies must be conducted to evaluate the success of in vitro assays under food environment conditions and to elucidate the antifungal mechanism of these strains.

Bacteriocins and other bioactive substances of probiotic lactobacilli as biological weapons against Neisseria gonorrhoeae.

In the search of new antimicrobial agents against Neisseria gonorrhoeae, the bacteriocins-producing probiotic lactobacilli deserve special attention. The inhibitory effects of biosubstances such as organic acids, hydrogen peroxide and each bacteriocin-like inhibitory substance (BLIS) L23 and L60 on the growth of different gonococcal strains were investigated. Different non-treated and treated cell-free supernatants of two probiotic lactobacilli containing these metabolites were used. The aims of this work were (i) to evaluate the antimicrobial activity of the biosubstances produced by two probiotic lactobacilli, estimating the proportion in which each of them is responsible for the inhibitory effect, (ii) to define their minimum inhibitory concentrations (MICs) and, (iii) to determine the potential interactions between these biosubstances against N. gonorrhoeae. The main antimicrobial metabolites were the BLIS-es L23 and L60 in comparison with other biosubstances. Proportionally, their contributions to the inhibition on the gonococcal growth were 87.28% and 80.66%, respectively. The MIC values of bacteriocins were promising since these substances, when diluted, showed considerable inhibitory activity for all gonococci. In the interaction between bacteriocins, 100% of synergism was found on the gonococcal growth. In summary, this study indicates that both L23 and L60 could potentially serve to design new bioproducts against N. gonorrhoeae.

Antifungal activity of two Lactobacillus strains with potential probiotic properties.

Aflatoxin (highly toxic and carcinogenic secondary metabolites produced by fungi) contamination is a serious problem worldwide. Modern agriculture and animal production systems need to use high-quality and mycotoxin-free feedstuffs. The use of microorganisms to preserve food has gained importance in recent years due to the demand for reduced use of chemical preservatives by consumers. Lactic acid bacteria are known to produce various antimicrobial compounds that are considered to be important in the biopreservation of food and feed. Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23 are producers of secondary metabolites, such as organic acids, bacteriocins and, in the case of L60, hydrogen peroxide. The antifungal activity of lactobacilli strains was determined by coculture with Aspergillus section Flavi strains by two qualitative and one quantitative methods. Both L23 and L60 completely inhibited the fungal growth of all aflatoxicogenic strains assayed. Aflatoxin B (1) production was reduced 95.7-99.8% with L60 and 27.5-100% with L23. Statistical analysis of the data revealed the influence of L60 and L23 on growth parameters and aflatoxin B (1) production. These results are important given that these aflatoxicogenic fungi are natural contaminants of feed used for animal production, and could be effectively controlled by Lactobacillus L60 and L23 strains with probiotic properties.

Lactobacillus rhamnosus L60, a potential probiotic isolated from the human vagina.

The vagina has been increasingly viewed as an "ecosystem" whose normal microflora help protect it from invading pathogens, including those that cause urinary tract infections and sexually transmitted diseases. We tested new strains of lactobacilli as potential probiotics for maintenance of urogenital tract health, as well as prevention and therapy of urogenital infections. A strain of lactobacilli isolated from the vagina of nonpregnant, healthy, premenopausal women was identified as Lactobacillus rhamnosus L60 by 16S rDNA sequence homology. L60 was evaluated for antimicrobial activity, in vitro antibiotic resistance, autoaggregation, surface hydrophobicity, co-aggregation with other bacterial species, hydrogen peroxide (H(2)O(2)) production, and bacterial adherence. It displayed a wide spectrum of antimicrobial activity against urogenital pathogens, and resistance to antibiotics commonly prescribed for infections caused by these pathogens. L60 produced H(2)O(2), adhered to vaginal epithelial cells, co-aggregated with Escherichia coli and Candida albicans, and displayed self-aggregation. In view of these characteristics, L60 is considered a potential probiotic, and will be further evaluated for preventive and therapeutic application locally in the vaginal tract.

Lactobacillus species isolated from the vagina: identification, hydrogen peroxide production and nonoxynol-9 resistance.

BACKGROUND: Lactic acid production is considered to be the major protection mechanism of lactobacilli against vaginal infections due to genital pathogens. Some species of Lactobacillus are also hydrogen peroxide (H(2)O(2)) producers. Nonoxynol-9 (N-9) is a nonionic detergent and is the active component of many spermicidal preparations. It immobilizes sperm by disrupting the cell membrane and is believed to act similarly on a number of bacteria and viruses. It is known that N-9 inhibits Lactobacilli in vitro at concentrations of 0.1% to 1%. PURPOSE: The present study was conducted to identify the species of Lactobacillus isolated from vaginal fluids of reproductive-age women and to characterize the H(2)O(2)-producing and N-9-resistant strains in an Argentine population. RESULTS: We identified Lactobacillus acidophilus, L. fermentum, L. gasseri, L. jensenii, L. casei subsp. casei, L. brevis and L. delbrueckii subsp. delbrueckii as the most frequent species. In this Argentine, South American population, 62% of women had H(2)O(2)-producing vaginal lactobacilli. We found a high number of sensitive strains. Sixty-two H(2)O(2)-producer strains were detected, 50 (80.6%) strains were sensitive to N-9 and 12 (19.4%) strains were resistant to the inhibitory effect of N-9. DISCUSSION: The vaginal microecologic findings are comparable to those found in other populations and suggest that (1) vaginal microecologic conditions are likely to be similar among Argentine women as in other countries, and (2) N-9 may have deleterious effects as in other populations.

Anticuerpos antimicroorganismos comunes en niños de diferentes edades / Microorganism antibodies common in children of ages different

Este trabajo tuvo por objeto investigar cualitativa y semicualitativamente Ac. contra una variedad de MO comunes: Pr; EC; Stp; Str; Kl y Ps en niños sanos, de clase social media alta, de la ciudad de Río Cuarto, Provincia de Córdoba (Argentina) y comparar sus títulos en relación a la edad. Para investigar los ac se utilizó la técnica de Inmunofluorescencia indirecta. De acuerdo con la edad de los niños se conforman cuatro Grupos. Los Ac. Anti: Pr, Kl y Ps fueron observados en una cantidad significativamente menor de niños en el Grupo I. La tendencia a encontrar estos Ac, en un número mayor de casos, está desplazada hacia los Grupos II, III y IV (niños mayores). La frecuencia de casos en la que se halló Ac. Anti: EC, Stp, Str fue similar en todos los grupos. Los Ac. antibacterianos, generalmente, estaban en concentraciones mayores (títulos más altos) en los Grupos II,III y IV. En nuestra causística se hallaron Ac. con especificidad y niveles definidos, con las variaciones cuali y cuantitativas que se demuestran. Estos hallazgos podrían atribuirse a la forma particular de respuestas de anticuerpos, inducidas por los MO que, sin duda, son influenciados por los hábitos personales, el ambiente y la edad de las personas. El estudio de los distintos Ac. contra MO resulta de interés en la mejor comprensión de la inmunidad individual y/o colectiva.

Anticuerpos antimicroorganismos comunes en niños de diferentes edades / Microorganism antibodies common in children of ages different

Este trabajo tuvo por objeto investigar cualitativa y semicualitativamente Ac. contra una variedad de MO comunes: Pr; EC; Stp; Str; Kl y Ps en niños sanos, de clase social media alta, de la ciudad de Río Cuarto, Provincia de Córdoba (Argentina) y comparar sus títulos en relación a la edad. Para investigar los ac se utilizó la técnica de Inmunofluorescencia indirecta. De acuerdo con la edad de los niños se conforman cuatro Grupos. Los Ac. Anti: Pr, Kl y Ps fueron observados en una cantidad significativamente menor de niños en el Grupo I. La tendencia a encontrar estos Ac, en un número mayor de casos, está desplazada hacia los Grupos II, III y IV (niños mayores). La frecuencia de casos en la que se halló Ac. Anti: EC, Stp, Str fue similar en todos los grupos. Los Ac. antibacterianos, generalmente, estaban en concentraciones mayores (títulos más altos) en los Grupos II,III y IV. En nuestra causística se hallaron Ac. con especificidad y niveles definidos, con las variaciones cuali y cuantitativas que se demuestran. Estos hallazgos podrían atribuirse a la forma particular de respuestas de anticuerpos, inducidas por los MO que, sin duda, son influenciados por los hábitos personales, el ambiente y la edad de las personas. El estudio de los distintos Ac. contra MO resulta de interés en la mejor comprensión de la inmunidad individual y/o colectiva. (AU)

Actividad hemolítica de Klebsiella pneumoniae sobre glóbulos rojos de conejo / Hemolytic activity of Klebsiella pneumoniae on rabbit erythrocytes

Se investigó la actividad hemolítica de Klebsiella pneumoniae, empleando cepas de referencia y cepas aisladas de infecciones. Todas las bacterias estudiadas resultaron hemolíticas en forma selectiva sobre eritrocitos de conejo. El frío intensificó el efecto lítico sobre los glóbulos rojos. Se estimó la capacidad hemolítica de las cepas mediante el diámetro de los halos producidos alrededor de orificios inoculados con 5 micron**l de suspensiones bacterianas. Cuando se realizó la detección de hemolisis en medios líquidos, sólo fue posible evidenciar lisis franca al tratar los cultivos con ciertos agentes reductores; de los cuales el más efectivo resultó el 2 mercaptoetanol. A la misma concentración los demás agentes químicos utilizados dieron menor porcentaje de eritrocitos lisados. La actividad hemolítica apareció en los cultivos durante la fase exponencial del crecimiento bacteriano y al cabo de 24 horas disminuyó al 33% del valor máximo alcanzado. Tanto la hemolisina cruda como la purificada reqirieron SH-activación y fueron inhibidas por suero

Nueva hemolisina oxígeno- lábil en Klebsiella pneumonial / New oxygen- labile hemolysin in Klebsiella pneumoniae

Se investigó la incidencia de hemolisinas en Klebsiella pneumoniae procedentes de cipas mantenidas en colección y de aislamientos a partir de materiales biológicos, encontrándose en el 100% de las cepas estudiadas actividad hemolítica selectiva sobre eritrocitos de conejo. Fue posible purificar dos fracciones hemolíticas a partir de los sobrenadantes de cultivo. Ambas result ron sulhidrilactivadas dependientes del tratamiento con agentes reductores y presentaron similitud con otras hemolisinas oxígeno-lábiles, en cuanto al mecanismo de acción e inhibidores. Mediante precipitación salina, filtración en gel Sephadex G-100, electroforesis en poliacrilamida y cromatografía en DEAE-Sephadex, se lograron separar dichas hemolisinas de proteínas contaminantes, una de las cuales tienen un efecto inhibitorio sobre la actividad hemolítica. Se alcanzaron porcentajes ejevados de recuperación, sobre todo en los pasos del proceso de purificación fundamentados en diferencias de densidad de carga eléctrica de las macromoléculas. Si bien la separación de las dos fracciones hemolíticas induce a pensar en estructuras químicas distintas por tener tamaño y carga eléctrica diferente, resulta sugestivo el hecho que comparten varias propiedades: activación de grupos sulfhidrilos, selectividad sobre eritrocitos de conejo, comport amiento frente al tratamiento térmico, pH óptimo, mecanismo de acción, inhidición por colesterol y cationes divalentes. En función de ello no se sugiere una denominación individual por el momento. Las hemolisinas purificadas constituyen el primer ejemplo de lisinas tiol-activadas en bacilos Gramnegativos

Nueva hemolisina oxígeno- lábil en Klebsiella pneumonial / New oxygen- labile hemolysin in Klebsiella pneumoniae

Se investigó la incidencia de hemolisinas en Klebsiella pneumoniae procedentes de cipas mantenidas en colección y de aislamientos a partir de materiales biológicos, encontrándose en el 100% de las cepas estudiadas actividad hemolítica selectiva sobre eritrocitos de conejo. Fue posible purificar dos fracciones hemolíticas a partir de los sobrenadantes de cultivo. Ambas result ron sulhidrilactivadas dependientes del tratamiento con agentes reductores y presentaron similitud con otras hemolisinas oxígeno-lábiles, en cuanto al mecanismo de acción e inhibidores. Mediante precipitación salina, filtración en gel Sephadex G-100, electroforesis en poliacrilamida y cromatografía en DEAE-Sephadex, se lograron separar dichas hemolisinas de proteínas contaminantes, una de las cuales tienen un efecto inhibitorio sobre la actividad hemolítica. Se alcanzaron porcentajes ejevados de recuperación, sobre todo en los pasos del proceso de purificación fundamentados en diferencias de densidad de carga eléctrica de las macromoléculas. Si bien la separación de las dos fracciones hemolíticas induce a pensar en estructuras químicas distintas por tener tamaño y carga eléctrica diferente, resulta sugestivo el hecho que comparten varias propiedades: activación de grupos sulfhidrilos, selectividad sobre eritrocitos de conejo, comport amiento frente al tratamiento térmico, pH óptimo, mecanismo de acción, inhidición por colesterol y cationes divalentes. En función de ello no se sugiere una denominación individual por el momento. Las hemolisinas purificadas constituyen el primer ejemplo de lisinas tiol-activadas en bacilos Gramnegativos (AU)