Low-cost culture media designed for biomass production of beneficial lactic acid bacteria for their inclusion in a formula to treat bovine reproductive infections.

As a first step for the use of probiotics in a formula for cattle, it is required to have available low-cost culture medium(s) and efficient production conditions for the growth of probiotic bacteria and high production of cell biomass. De Man-Rogosa-Sharpe medium, used frequently for lactic acid bacteria (LAB) contains adequate ingredients for their growth but is very expensive for industrial application. The nutrients required for LAB growth are strain-dependent. In this work, traditional culture media were evaluated omitting and/or modifying ingredients in their composition, as carbon or nitrogen source, on the basis of their low-cost industrial waste, to select those supporting the most efficient growth. The results showed that the formulation of culture media containing fructose (0.5%) and molasses (1.0%) was better for the growth and production of cell biomass for all the strains assayed, except Lactobacillus gasseri CRL1421 growing in 1.5% corn syrup. FM902 yeast extract at concentrations between 1.5% and 2.5% was the most adequate for most of the strains. The LAB grown in the designed media maintained the beneficial properties for which they selected. The use of the culture media designed to produce biomass decrease production costs, which is an important step for the feasible industrial production of probiotic pharmaceuticals.

Functional and safety characterization of beneficial vaginal lactic acid bacteria for the design of vaginal hygiene products.

AIMS: The aim was to complement the evaluation of functional and safety properties of beneficial vaginal lactic acid bacteria to select the most adequate strains as potential probiotics for their inclusion in a vaginal probiotic formula. METHODS AND RESULTS: Twenty-four beneficial vaginal lactic acid bacteria strains previously isolated from healthy women were characterized by applying phenotypical and genetic techniques. The biofilm formation and exopolysaccharide production by the different strains showed a strong influence of the growth medium on the expression of these properties. The evaluated strains evidenced different antibiotic susceptibility patterns by phenotypic tests, while the resistance genes were not always correlated with the phenotypic profiles. None of the evaluated strains showed lecithinase, gelatinase, or ß-hemolytic activity. Based on the results obtained, Lactobacillus gasseri CRL 1320 and Lacticaseibacillus rhamnosus CRL 1332 were selected to advance in the design of the products. CONCLUSIONS: A wide variability in the properties expressed by beneficial vaginal lactic acid bacteria strains were shown, evidencing a strain-specific and independent behaviour of the species/metabolic group where classified. The selection criteria applied provided a valuable tool for the selection of the best vaginal lactobacilli strains for their inclusion in formulas for vaginal application. SIGNIFICANCE AND IMPACT OF THE STUDY: Since probiotics are an important tool for improving human health, the designed formula are an excellent alternative to reconstitute the vaginal microbiota and prevent/treat urogenital tract infections in women. The results obtained contribute to deepen the characterization of homologous beneficial vaginal lactic acid bacteria and advance in the design of vaginal probiotic products.

Administration of probiotic lactic acid bacteria to modulate fecal microbiome in feedlot cattle.

Modulation of animal gut microbiota is a prominent function of probiotics to improve the health and performance of livestock. In this study, a large-scale survey to evaluate the effect of lactic acid bacteria probiotics on shaping the fecal bacterial community structure of feedlot cattle during three experimental periods of the fattening cycle (163 days) was performed. A commercial feedlot located in northwestern Argentina was enrolled with cattle fed mixed rations (forage and increasing grain diet) and a convenience-experimental design was conducted. A pen (n = 21 animals) was assigned to each experimental group that received probiotics during three different periods. Groups of n = 7 animals were sampled at 40, 104 and 163 days and these samples were then pooled to one, thus giving a total of 34 samples that were subjected to high-throughput sequencing. The microbial diversity of fecal samples was significantly affected (p < 0.05) by the administration period compared with probiotic group supplementation. Even though, the three experimental periods of probiotic administration induced changes in the relative abundance of the most representative bacterial communities, the fecal microbiome of samples was dominated by the Firmicutes (72-98%) and Actinobacteria (0.8-27%) phyla, while a lower abundance of Bacteroidetes (0.08-4.2%) was present. Probiotics were able to modulate the fecal microbiota with a convergence of Clostridiaceae, Lachnospiraceae, Ruminococcaceae and Bifidobacteriaceae associated with health and growth benefits as core microbiome members. Metabolic functional prediction comparing three experimental administration periods (40, 104 and 163 days) showed an enrichment of metabolic pathways related to complex plant-derived polysaccharide digestion as well as amino acids and derivatives during the first 40 days of probiotic supplementation. Genomic-based knowledge on the benefits of autochthonous probiotics on cattle gastrointestinal tract (GIT) microbiota composition and functions will contribute to their selection as antibiotic alternatives for commercial feedlot.

Probiotic lactobacilli in formulas and hygiene products for the health of the urogenital tract.

Lactobacilli are the predominant microorganisms of the healthy human vagina. A novel alternative for the prevention and treatment of female urogenital tract infections (UGTI) is the inclusion of these microorganisms as active pharmaceutical ingredients in probiotic formulas, and more recently in female hygienic products. Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." A list of requirements must be considered during the development of probiotic product/formula for the female urogenital tract (UGT). This review aims to resume the requirements, probiotic characteristics, and clinical trial applied to determine the effect of probiotic and potentially probiotic strains on different woman's physiological and pathological conditions, and in preterm birth prevention. A revision of female hygienic products available in the world market is included, together with novel studies applying nanotechnology for Lactobacillus incorporation in hygienic products. Further studies and well-designed clinical trials are urgently required to complement the current knowledge and applications of probiotics in the female UGT. The use of probiotic formulas and products will improve and restore the ecological equilibrium of the UGT microbiome to prevent and treat UGTI in women under different conditions.

Safety, environmental and technological characterization of beneficial autochthonous lactic bacteria, and their vaginal administration to pregnant cows for the design of homologous multi-strain probiotic formulas.

In recent years, veterinary probiotic formulations constitute an interesting alternative to the use of antibiotics in animals for human consumption, but beneficial microorganisms must meet certain requirements to be included in these products. The objective of this work was to evaluate the safety and innocuity of beneficial autochthonous lactic bacteria (BALB) as well as to determine their beneficial, environmental, and technological characterization. Antibiotic resistance was assayed using phenotypic and genotypic methodology. A bovine vaginal fluid simulated medium (MSBVF) was designed where growth, pH changes, and expression of beneficial characteristics of lactic bacteria were evaluated; additionally, the optimal culture conditions in commercial media were determined in order to obtain the highest biomass production of the strains. Finally, the best strains were lyophilized and administered intravaginally to pregnant cows and their permanence in the vagina and adverse effects were evaluated. The results show that most of the strains were resistant to vancomycin, tetracycline, and streptomycin, with a high sensitivity to ampicillin, gentamicin, and clindamycin. The strains evaluated did not show gelatinase or hyaluronidase activity; however, 11 strains produced α-type hemolysis. The optimal growth of the microorganism was obtained in MRS broth, under slight agitation and without pH control. The strains grown in the MSBVF grew well and maintained the probiotic properties. Animals treated with probiotics bacteria did not show systemic or local inflammation. These strains can be included in a probiotic veterinary product to be applied to different bovine mucosa.

Productos elaborados con verdolaga (Portulaca Olerácea L. ): valoración nutricional, conocimiento, características organolépticas, aceptabilidad y satisfacción / Products made whith verdolaga (Portulaca Oleracea L. ): nutritional assessment, knowledge, organeleptic characteristics, acceptance and satisfaction

Introducción: Portulaca oleracea, conocida como verdolaga y considerada culturalmente una maleza, presenta múltiples propiedades benéficas que permiten emplearla como alimento debido a su fácil acceso y bajo costo. No se detectan muchos estudios sobre el tema. Objetivos: elaborar productos alimenticios empleando verdolaga como materia prima, con características organolépticas adecuadas; calcular su valor nutricional y determinar el porcentaje de ingesta diaria recomendada (IDR) que cubre; identificar el nivel de conocimiento sobre la verdolaga y sus propiedades nutricionales; determinar las características organolépticas de los alimentos elaborados, el grado de aceptabilidad y satisfacción en la población en estudio. Materiales y métodos: estudio de tipo descriptivo, no experimental y experimental (pre experimento), de corte transversal. Se elaboraron tres preparaciones con verdolaga: sopa (S), tortilla (T) y ensalada (E), y se sometieron a degustación. Resultados: el valor calórico de las preparaciones por porción fue: S: 24,2 kilocalorías (kcal)/200 g, T: 129,2 kcal/200 g, y E: 116,16 kcal/150 g. Los porcentajes de IDR que cubre una porción de cada alimento fueron: S: 1,21% kcal, 1,31% hidratos de carbono, 1,96% proteínas, 0,54% lípidos; T: 6,43% kcal, 3,48% hidratos de carbono, 9,54% proteínas, 11,81% lípidos, y E: 5,8% kcal, 5,06% hidratos de carbono, 4,28% proteínas y 8,6% lípidos. Estos valores se calcularon en base a los valores diarios de referencia de nutrientes (VDR) de declaración obligatoria que son: 2.000 kcal, 300 g de hidratos de carbono, 75 g de proteínas y 55 g de lípidos para un grupo de hombres y mujeres adultos de entre 18 y 60 años de edad. La muestra estuvo formada por 50 personas, de 42 años edad promedio, 58% femenino y 42% masculino, 40% con estudios secundarios, 34% universitarios (N=17), 18% terciarios (N=9) y 8% primarios. El 76% presentó un conocimiento bajo sobre la verdolaga y sus propiedades nutricionales. Las elaboraciones fueron satisfactorias para el 82% de los encuestados y aceptadas por el 90% Conclusiones: en base a las propiedades nutricionales que se sugieren y su alta versatilidad para incorporar en diversas recetas, la verdolaga se presenta como una excelente alternativa para la alimentación humana

Introduction: portulaca oleracea, known as purslane (verdolaga), culturally considered an undergrowth, has multiple beneficial properties allowing to be used as a food, due to its easy access and low cost. There are not many studies on the subject. Objectives: the objective of present work was to prepare food products using purslane as raw material, with adequate organoleptic characteristics. To calculate their nutritional value and to determine the recommended dietary allowance (RDA) percentage they cover. Also, to identify the level of knowledge that a certain population has about purslane and its nutritional properties, and to define the organoleptic characteristics of the foods elaborated, evaluating the degree of acceptability and satisfaction in the population under study. Materials and methods: the study was descriptive, nonexperimental and experimental (pre-experiment), cross-sectional. Three preparations with purslane were elaborated and subjected to tasting: soup (S), omelette (O) and salad (E). Results: tthe caloric value per serving of the preparations was S: 24.2 kcal/200 g; O: 129.2 Kcal/200 g, and E: 116.16 kcal/150 g. The percentages of RDA covered by a serving of each food were: S: 1.21% kcal, 1.31% carbohydrates, 1.96% proteins, 0.54% fats; T: 6.43% kcal, 3.48% carbohydrates, 9.54% proteins, 1.81% fats, and E: 5.8% kcal, 5.06% carbohydrates, 4.28% proteins and 8.6% fats. The values were calculated on referred to the recommended daily intake (RDI), which are: 2,000 kcal, 300 g carbohydrates, 75 g proteins y 55 g fats, defined to a group of adult women and men between 18 and 60 years old. The sample included 50 people with 42 years average age, 58% female and 42%male, 40% with secondary studies, 34% university (N=17), 18% tertiary (N=9) and 8% primary. 76% of the participants presented a low knowledge about purslane and its nutritional properties. The preparations were satisfactory for 82% of the participants and accepted by 90%. Conclusions: due to the suggested nutritional properties and its versatility for its incorporation in various recipes, purslane can be indicated as an excellent alternative for human consumption

Immobilization of vaginal Lactobacillus in polymeric nanofibers for its incorporation in vaginal probiotic products.

Probiotic products require high number of viable and active microorganisms during storage. In this work, the survival of human vaginal Lactobacillus gasseri CRL1320 and Lactobacillus rhamnosus CRL1332 after nanofiber-immobilization by electrospinning with polyvinyl-alcohol, and during storage was evaluated. The optimization of bacterial immobilization and storage conditions using bioprotectors (skim milk-lactose and glycerol) and oxygen-excluding packaging was carried out, compared with lyophilization. After electrospinning, a higher survival rate of L. rhamnosus (93%) compared to L. gasseri (84%) was obtained in nanofibers, with high viable cells (>107 colony-forming unit/g) of the two probiotics in nanofibers stored at -20°C up to 14 days. The storage in oxygen-excluding packaging was an excellent strategy to extend the shelf-life of L. rhamnosus (up to 1.7 × 108 CFU/g) in nanofibers stored at 4°C during 360 days, with no addition of bioprotectives, resulting similar to freeze-dried-cells. L. rhamnosus was successfully incorporated into polymeric hydrophilic nanofibers with a mean diameter of 95 nm. The composite materials were characterized in terms of morphology, and their physicochemical and thermal properties assessed. Nanofiber-immobilized L. rhamnosus cells maintained the inhibition to urogenital pathogens. Thus, polymeric nanofiber-immobilized L. rhamnosus CRL1332 can be included in vaginal probiotic products to prevent or treat urogenital infections.

Screening of autochthonous vaginal beneficial lactobacilli strains by their growth at high temperatures for technological applications.

The pharmaceutical industry shows an emerging interest in formulas that contain live and beneficial microorganisms, also known as probiotics or pharmabiotics, which in many cases, are host-specific. The resistance to higher temperature is an essential feature of these microorganisms when working on the design of products for vaginal formula. In order to obtain a high number of viable cells and a prolonged shelf life in the designed product, it is required to apply technological procedures using high temperatures or abrupt changes of them, which result in conditions that are different from the optimal growth temperature and can affect the metabolic capabilities of the bacteria when administered to the host in order to reestablish the ecological mucosa. The aim of this work was to evaluate the behavior of 30 different species and strains of autochthonous beneficial vaginal lactobacilli (BVL) when exposed to high temperatures, determine their survival capabilities and analyze their pre-adaptation to those temperatures, in order that they still maintain their viability after technological processes and further conservation. BVL were exhibited to temperatures higher than optimal, with the purpose of evaluating their growth kinetics and parameters. Later, they were exposed to higher temperatures, and then, returned to their optimal, to determine if they were able to grow again. The strains that showed higher resistance were selected, and their viability and beneficial properties studied further. The growth kinetics of strains exposed to higher temperatures showed different patterns, which provided evidence that the thermal adaptation is strain-dependent and is not related to any particular species and/or metabolic group in which the strains were taxonomically classified. The pre-adaptive step allowed the growth of some of the strains, preserving their viability and probiotic properties after the high temperatures were applied. The results shows that BVL can be exposed to high temperatures used in different technological processes that are applied for pharmabiotic formulations, such as spray dried or vacuum rotary evaporation, and/or during the conservation period. The results obtained indicate that some specific BVL strains resist high temperatures and grow afterwards at optimal conditions.

Erratum: Fukuyama, K., et al. Evaluation of the Immunomodulatory Ability of Lactic Acid Bacteria Isolated from Feedlot Cattle Against Mastitis Using a Bovine Mammary Epithelial Cells In Vitro Assay. Pathogens 2020, 9, 410.

The authors would like to make the following corrections about the published paper [...].

Biosurfactant from vaginal Lactobacillus crispatus BC1 as a promising agent to interfere with Candida adhesion.

BACKGROUND: Lactobacillus spp. dominating the vaginal microbiota of healthy women contribute to the prevention of urogenital and sexually transmitted infections. Their protective role in the vagina can be mediated by Lactobacillus cells themselves, metabolites or bacterial components, able to interfere with pathogen adhesion and infectivity. Vulvovaginal candidiasis (VVC) is a common genital infection, caused by the overgrowth of opportunistic Candida spp. including C. albicans, C. glabrata, C. krusei and C. tropicalis. Azole antifungal drugs are not always efficient in resolving VVC and preventing recurrent infections, thus alternative anti-Candida agents based on vaginal probiotics have gained more importance. The present work aims to chemically characterize the biosurfactant (BS) isolated from a vaginal Lactobacillus crispatus strain, L. crispatus BC1, and to investigate its safety and antiadhesive/antimicrobial activity against Candida spp., employing in vitro and in vivo assays. RESULTS: BS isolated from vaginal L. crispatus BC1 was characterised as non-homogeneous lipopeptide molecules with a critical micellar concentration value of 2 mg/mL, and good emulsification and mucoadhesive properties. At 1.25 mg/mL, the BS was not cytotoxic and reduced Candida strains' ability to adhere to human cervical epithelial cells, mainly by exclusion mechanism. Moreover, intravaginal (i.va.) inoculation of BS in a murine experimental model was safe and did not perturb vaginal cytology, histology and cultivable vaginal microbiota. In the case of i.va. challenge of mice with C. albicans, BS was able to reduce leukocyte influx. CONCLUSIONS: These results indicate that BS from vaginal L. crispatus BC1 is able to interfere with Candida adhesion in vitro and in vivo, and suggest its potential as a preventive agent to reduce mucosal damage occasioned by Candida during VVC.

Milk Microbiota: What Are We Exactly Talking About?

The development of powerful sequencing techniques has allowed, albeit with some biases, the identification and inventory of complex microbial communities that inhabit different body sites or body fluids, some of which were previously considered sterile. Notably, milk is now considered to host a complex microbial community with great diversity. Milk microbiota is now well documented in various hosts. Based on the growing literature on this microbial community, we address here the question of what milk microbiota is. We summarize and compare the microbial composition of milk in humans and in ruminants and address the existence of a putative core milk microbiota. We discuss the factors that contribute to shape the milk microbiota or affect its composition, including host and environmental factors as well as methodological factors, such as the sampling and sequencing techniques, which likely introduce distortion in milk microbiota analysis. The roles that milk microbiota are likely to play in the mother and offspring physiology and health are presented together with recent data on the hypothesis of an enteromammary pathway. At last, this fascinating field raises a series of questions, which are listed and commented here and which open new research avenues.

Anti-Candida activity of beneficial vaginal lactobacilli in in vitro assays and in a murine experimental model.

Vulvovaginal candidiasis (VVC) is one of the most frequent infections affecting women worldwide. Healthy vaginal microbiota is dominated by lactobacilli, which form a strong defense line against pathogens. In this work, in vitro antimicrobial properties of thirty vaginal Lactobacillus strains were evaluated against eleven Candida vaginal clinical isolates, employing three different methods. Also, the effect of intravaginal (i.va.) administrations (preventive, therapeutic and preventive-therapeutic) of L. reuteri CRL1324 or L. rhamnosus CRL1332 strains against the i.va. challenge with Candida albicans C2 (C.a.) was evaluated in a murine experimental model. From the results of agar overlay and liquid medium assays the selected lactobacilli strains have shown to inhibit the growth of at least one Candida strain. The inhibition was mainly due to the effect of organic acids. Anti-Candida activity was not evidenced in the agar plate diffusion method. In the experimental murine model, only preventive-therapeutic administration of both lactobacilli was able to significantly reduce viable C.a. numbers recovered in vaginal washes and the leukocyte influx induced by the fungi. In conclusion, lactobacilli exhibited in vitro and in vivo antimicrobial effects on Candida, suggesting that they could be promising candidates for protection against VVC.

Kipi crudo: accion del jugo de limón, aceptabilidad satisfacción y preferencia en una población de San Fernando del Valle de Catamarca / Raw kipi: action of lemon juice, acceptability, satisfaction and preference in a population in San Fernando del Valle de Catamarca

Introducción: El Kebbe Naihe o kipi crudo es un producto cárneo que tiene como ingredientes la carne bovina u ovina molida y trigo burgol, condimentos y especias y se consume crudo. La carne es el vehículo de microorganismos que causan brotes de enfermedades, infecciones e intoxicaciones microbianas y resulta riesgosa su incorporación, dada la frecuencia con la que se pueden aislar bacterias patógenas a partir del producto crudo. El uso de diferentes sustancias controla el número de microorganismos en preparaciones alimenticias. Objetivos: Determinar el efecto del jugo de limón sobre los microorganismos presentes en el kipi crudo, analizar la aceptabilidad y satisfacción del kipi crudo con limón, y la preferencia del kipi crudo con limón respecto del kipi crudo sin limón. Metodología: Estudio de tipo descriptivo, correlacional y explicativo, y con un diseño en dos etapas, una etapa experimental- experimento puro y en otra etapa experimental-preexperimento. Resultados: Los análisis microbiológicos indicaron que en el momento de elaboración y a las 6, 12 y 24hs, el número de microorganismos fue menor en el kipi crudo tratado con limón, comparado con el que no recibió este tratamiento. Asimismo, el jugo de limón disminuyó el número de bacterias del kipi crudo a medida que el tiempo de exposición a este fue mayor. El kipi crudo con jugo de limón resultó ser más aceptado, satisfactorio y preferido por la población no árabe, en comparación con la población árabe. El kipi crudo es un producto apto para el consumo desde el punto de vista microbiológico si se consume antes de las 6hs posteriores a su elaboración, lo cual permite a la población árabe continuar consumiendo este plato sin el agregado de limón. Conclusión: Es posible, para quienes les resulta agradable el kipi crudo con limón, incrementar su inocuidad mediante el agregado de esta sustancia natural

Introduction: Kebbe Naihe or raw kipi is a meat-derived food, which has bovine or ovine ground meat and wheat burgol, condiments and spices as ingredients, and is consumed in raw state. Meat is the vehicle of microorganisms that cause diseases outbreaks, infections and microbial poisoning and its incorporation is risky given the frequency of isolation of pathogenic bacteria from raw products. The use of different substances controls the number of microorganisms in food preparations. Objectives: to determine the effect of lemon juice on the microorganisms present in raw kipi, to analyze the acceptability and satisfaction of raw kipi with lemon, and the preference of raw kipi with lemon compared to raw kipi without lemon. Methodology: Descriptive, correlational and explanatory type study, with a two-stage design, a pure experimental-experimental stage and pre-experiment in another stage. Results: The microbiological analysis indicated that at the time of elaboration and at 6, 12 and 24 hours, the number of microorganisms was lower in the raw kipi treated with lemon, compared to the one that did not receive this treatment. The lemon juice decreased the number of bacteria of the raw kipi at higher exposure times. The raw kipi with lemon juice resulted highly accepted, satisfactory and preferred by the non- Arabic population, compared to the Arabic group. Raw kipi is a suitable product for consumption from the microbiological point of view only if consumed before 6 hours after its preparation, which allows the Arabic population to continue consuming this plate without the addition of lemon. Conclusion: It is possible, for those who like the raw kipi with lemon, to increase its safety by adding lemon juice, a natural substance

In vitro effect of vaginal lactobacilli on the growth and adhesion abilities of uropathogenic Escherichia coli.

Escherichia coli is one of the main causes of uncomplicated urinary tract infections and responsible of vaginal infections. Lactobacilli can inhibit this pathogen by the production of antimicrobial substances as organic acids, hydrogen peroxide and/or bacteriocins. The aim of this work was to study the effects of beneficial vaginal lactobacilli on E. coli through in vitro experiments. The inhibitory activity of three vaginal Lactobacillus strains against E. coli was assessed using the agar plate diffusion. Moreover, the effect of Lactobacillus reuteri CRL (Centro de Referencia para Lactobacilos Culture Collection) 1324 on the adhesion and internalization capabilities of E. coli was studied on HeLa cells. Two Lactobacillus strains inhibited the growth of the pathogens by production of organic acids. L. reuteri CRL 1324 reduced the adhesion and internalization of E. coli 275 into HeLa cells. The results obtained suggest that L. reuteri CRL 1324 can be considered as a probiotic candidate for further in vivo studies for the prevention or treatment of urinary tract infections caused by E. coli.

Biofilms of vaginal Lactobacillus reuteri CRL 1324 and Lactobacillus rhamnosus CRL 1332: kinetics of formation and matrix characterization.

Adhesion and biofilm formation are strain properties that reportedly contribute to the permanence of lactobacilli in the human vagina. The kinetics of biofilm formation and the chemical nature of the biofilm matrix formed by Lactobacillus reuteri CRL (Centro de Referencia para Lactobacilos Culture Collection) 1324 and Lactobacillus rhamnosus CRL 1332, vaginal beneficial strains, were evaluated in this work. Crystal violet-stained microplate assay and techniques of epifluorescence, electron and confocal microscopy were applied. The highest density and complexity of biofilms of both vaginal lactobacilli were observed at 72 h of incubation. Protease, proteinase K, α-chymotrypsin and trypsin treatments efficiently detached L. reuteri CRL 1324 biofilm that was also partially affected by α-amylase. However, L. rhamnosus CRL 1332 biofilm was slightly affected by protease, proteinase K and α-amylase. Confocal microscopy revealed greater amount of polysaccharides in L. rhamnosus CRL 1332 biofilm matrix than in L. reuteri CRL 1324 biofilm matrix. The results indicate that proteins are one of the main components of the L. reuteri CRL 1324 biofilm, while the biofilm matrix of L. rhamnosus CRL 1332 is composed of carbohydrates and proteins. The results obtained support the knowledge, understanding and characterization of two biofilm-forming vaginal Lactobacillus strains.

Immunomodulation of Lactobacillus reuteri CRL1324 on Group B Streptococcus Vaginal Colonization in a Murine Experimental Model.

PROBLEM: Maternal Group B Streptococcus (GBS) colonization is a risk factor for infectious disease in newborns. One promising strategy is the modulation of vaginal defense to increase the host's ability to combat infection. METHOD OF STUDY: The effect of intravaginal (i.va.) Lactobacillus reuteri CRL1324 inoculation on different immune cell populations, cytokines, and immunoglobulin isotypes in a murine model of GBS vaginal colonization was evaluated. RESULTS: Seven i.va. inoculations of L. reuteri CRL1324 previous to GBS challenge showed an immunomodulatory effect on the cells and mediators of innate immunity, decreasing the number of neutrophils induced by the pathogen and increasing the activated macrophage population. Moreover, increases in B lymphocytes and IgA and IgG subclasses were observed in mice inoculated with L. reuteri CRL1324 and then challenged with GBS. CONCLUSION: Lactobacillus reuteri CRL1324 shows a protective effect against GBS colonization that could be mediated by the modulation of the immune response.

Design of a beneficial product for newborn calves by combining Lactobacilli, minerals, and vitamins.

Diarrhea is one of the most frequent diseases affecting newborn calves in intensive systems. Several strategies were proposed to protect and improve health, such as probiotics. This work was directed to design a product containing freeze-dried bacteria, vitamins, and minerals, as well as to optimize conditions with lyoprotectors, combine strains and add vitamins, minerals, and inulin to the product. The lyoprotectors were milk, milk-whey, and actose, and products were stored for 6 months at 4°C. Combined bacteria were freeze-dried in milk and the final products were added with minerals, vitamins, and insulin. The viable cells were determined by the plate count assay and antibiotic profiles to differentiate strains. Lactobacillus johnsonii CRL1693, L. murinus CRL1695, L. mucosae CRL1696, L. salivarius CRL1702, L. amylovorus CRL1697, and Enterococcus faecium CRL1703 were evaluated. The optimal conditions were different for each strain. Milk and milk whey maintained the viability during the process and storage after 6 months for most of the strains, except for L. johnsonii. Lactose did not improve cell's recovery. L. murinus was viable for 6 months in all the conditions, with similar results in enterococci. In strains combined before freeze-dried, the viability decreased deeply, showing that one-step process with bacteria mixtures, vitamins, and minerals were not adequate. Freeze-dried resistance depends on each strain and must be lyophilized individually.

Encapsulation and subsequent freeze-drying of Lactobacillus reuteri CRL 1324 for its potential inclusion in vaginal probiotic formulations.

Probiotic formulations must include a high number of viable and active microorganisms. In this work, the survival of human vaginal Lactobacillus reuteri CRL 1324 during encapsulation, lyophilization and storage, and the activity of encapsulated and/or freeze-dried bacterial cells were evaluated. Extrusion-ionic gelation technique was applied to encapsulate L. reuteri CRL 1324, using xanthan and gellan. Encapsulated and free bacterial cells were freeze-dried with or without lactose and skim milk as lyoprotectors. The different systems obtained were stored at room temperature and at 4°C for 150days. The following determinations were performed: L. reuteri CRL 1324 viability, microorganism released from capsules, survival in a medium simulating the vaginal fluid and maintenance of beneficial properties (growth inhibition of opportunistic pathogenic Streptococcus agalactiae NH 17 and biofilm formation). L. reuteri CRL 1324 encapsulation was efficient, allowing the recovery of a high number of entrapped lactobacilli. The survival of encapsulated L. reuteri during lyophilization and storage was significantly higher in the presence of lyoprotectors. At the end of storage, the highest numbers of viable cells were obtained in free or encapsulated cells freeze-dried with lyoprotectors, stored at 4°C. Encapsulated and/or lyophilized L. reuteri cells maintained their viability in simulated vaginal fluid as well as the ability to inhibit S. agalactiae NH 17 growth and to form biofilm. Encapsulated and freeze-dried L. reuteri CRL 1324 can be included in a suitable pharmaceutical form for vaginal application to prevent or treat urogenital infections in women.

Profiles and technological requirements of urogenital probiotics.

Probiotics, defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, are considered a valid and novel alternative for the prevention and treatment of female urogenital tract infections. Lactobacilli, the predominant microorganisms of the healthy human vaginal microbiome, can be included as active pharmaceutical ingredients in probiotics products. Several requirements must be considered or criteria fulfilled during the development of a probiotic product or formula for the female urogenital tract. This review deals with the main selection criteria for urogenital probiotic microorganisms: host specificity, potential beneficial properties, functional specifications, technological characteristics and clinical trials used to test their effect on certain physiological and pathological conditions. Further studies are required to complement the current knowledge and support the clinical applications of probiotics in the urogenital tract. This therapy will allow the restoration of the ecological equilibrium of the urogenital tract microbiome as well as the recovery of the sexual and reproductive health of women.

Design of novel urogenital pharmabiotic formulations containing lactobacilli, salivaricin CRL 1328 and non-microbial compounds with different functionalities.

CONTEXT: The administration of pharmabiotics is a promising alternative to antimicrobial drugs for the treatment and/or prevention of female urogenital infections. OBJECTIVE: To design pharmabiotic formulations including bioactive ingredients of microbial origin combined with non-microbial substances and then to evaluate the stability of the combinations during freeze-drying and storage. MATERIALS AND METHODS: Different formulations including Lactobacillus gasseri CRL 1263, Lactobacillus salivarius CRL 1328, salivaricin CRL 1328 (a bacteriocin) and non-microbial compounds (lactose, inulin and ascorbic acid) were assayed, and the ingredients were freeze-dried together or separately. The formulations were stored in gelatin capsules at 4 °C for 360 d. RESULTS: The viability of lactobacilli was affected to different extents depending on the strains and on the formulations assayed. L. salivarius and ascorbic acid were successfully combined only after the freeze-drying process. Salivaricin activity was not detected in formulations containing L. gasseri. However, when combined with ascorbic acid, lactose, inulin or L. salivarius, the bacteriocin maintained its activity for 360 d. The selected microorganisms proved to be compatible for their inclusion in multi-strain formulations together with lactose, inulin and ascorbic acid. Salivaricin could be included only in a L. salivarius CRL 1328 single-strain formulation together with non-microbial substances. CONCLUSIONS: This study provides new insights into the design of urogenital pharmabiotics combining beneficial lactobacilli, salivaricin CRL 1328 and compounds with different functionalities.