Microencapsulation of Probiotics with Soy Protein Isolate and Alginate for the Poultry Industry.

Many probiotic products, with properly selected microorganisms, may not be effective for the intended purpose due to the low tolerance of microorganisms to gastrointestinal digestion. The microencapsulation seems to be one of the most promising techniques to protect probiotics against adverse environmental conditions. Therefore, the aim of this work was the design of soy protein isolate-alginate microcapsules for the encapsulation of probiotics for the poultry industry by the water-in-oil emulsion technique. To this end, the strain Ligilactobacillus salivarius CRL2217, with the ability to bind wheat germ agglutinin (WGA) on its surface and protect intestinal epithelial cells from the cytotoxicity of the glycoprotein, was used as model microorganism. Several parameters were varied in order to find the better conditions for microencapsulation: oil source and nature, SPI and sodium alginate concentration, stirring equipment and time for emulsion formation, CaCl2 concentration, and absence or presence of stirring after the addition of the CaCl2 solution. The survival of entrapped cells to a simulated gastric digestion and their survival and release during simulated intestinal digestion were also investigated. The obtained particles effectively protected L. salivarius CRL2217 from the proteolytic activity and low pH present in the gastric environment. Besides, their content was released in contact with a simulated intestinal juice, as viable counts and binding of WGA after a simulated intestinal digestion revealed. This work paves the way for the design of probiotic supplements for poultry including gastrointestinal digestion-susceptible bacteria.

Characterization of lactic acid bacteria isolated from the poultry intestinal environment with anti-Salmonella activity in vitro.

The purpose of this research was the genotypic identification of lactic acid bacteria (LAB), isolated from the gastrointestinal tract (GIT) of healthy adult birds, and the study of their safety regarding antibiotic resistance, physiological and functional properties involved in the colonization of the GIT of poultry, and Salmonella exclusion, as members of a potential mixed probiotic supplement for poultry. The nucleotidic sequence from Lactobacillus crispatus P1, L. animalis L3, and Enterococcus faecium CRL 1385 (ex-J96) showed 100, 99.8, and 99.3% identity with L. crispatus DSM 20584 T, Ligilactobacillus salivarius ATCC 11741 T, and E. faecium ATCC 19434 T, respectively. These strains showed no resistance to relevant antibiotics usually administered to animals proposed by the European Food Safety Authority. They could endure the detrimental conditions of the gastrointestinal tract (pH 2.6 and oxgall 0.1 and 0.4% w/v). In an ex vivo assay, the LAB showed high adherence to the three sections of the GIT, reaching values higher than 70%. The adhesion to mucus was strain-dependent: L. crispatus CRL 1453 evidenced the highest adhesion (> 19%) while Lig. salivarius subsp. salivarius CRL 1417 and E. faecium CRL 1385 adhered to a lower extent (> 9 and 2%, respectively). Moreover, the LAB elicited remarkable anti-Salmonella activity, taking into account that they could inhibit elevated counts of different Salmonella serovars, especially the host-specific serovars S. Gallinarum and S. Pullorum (up to 8 log CFU/mL decrease in Salmonella counts).

Growth studies of dominant lactic acid bacteria in orange juice and selection of strains to ferment citric fruit juices with probiotic potential.

The study aimed to evaluate the ability of dominant lactic acid bacteria (LAB) in orange juice to growth on N-depleted MRS medium supplemented or not with cysteine (mMRS), then to select the most nutritionally promising strains for growth assays in the food matrix and evaluation of beneficial attributes for fruit juice fermentation. Levilactobacillus brevis and Lactiplantibacillus plantarum were dominant species among the total of 103 LAB isolates as confirmed by multiplex PCR and/or 16 s rDNA sequence analysis. Based on growing lower than 20% and higher than 70% in mMRS (1.0 g/l meat extract, without peptone and yeast extract) with and without cysteine requirement, one L. brevis (JNB23) and two L. plantarum (JNB21 and JNB25) were selected. These bacteria and the L. plantarum strains N4 and N8 (previously isolated from oranges peel) when inoculated in orange juice grew up to 1.0 log cfu/ml for 24 h incubation at 30 °C and mainly produced lactic acid, with strains JNB25 and JNB23 reaching the highest and lowest cell densities in agreement with their nutritional exigency. In addition, all L. plantarum strains exhibited antagonistic activity against the majority of tested bacterial pathogens (in opposition to L. brevis), ability to grow or survive to pH 3.0 for 3 h, to grow with 0.5% sodium taurocholate, and a decrease after simulated gastrointestinal digestion assay which did not exceed 1.0 or 2.0 log units, depending on the strain. Thus, autochthonous L. plantarum strains with ability for overcoming nutritional limitations and beneficial attributes are promising candidates for further investigations as novel probiotic and/or preservative starters to ferment citric fruit juices.

Ferulic Acid Esterase Producing Lactobacillus johnsonii from Goat Feces as Corn Silage Inoculants.

Ferulic acid esterase (FAE+)-producing lactobacilli are being studied as silage inoculants due to their potential of increasing forage fiber digestibility. In this work, three FAE+ Lactobacillus (L.) johnsonii strains were isolated from caprine feces and characterized according to their potential probiotic characteristics and as silage inoculants. Limosilactobacillus fermentum CRL1446, a human probiotic isolated from goat cheese, was also included in the experiments as a potential silage inoculant. FAE activity quantification, probiotic characterization, and growth in maize aqueous extract indicated that L. johnsonii ETC187 might have a better inoculant and probiotic aptitude. Nevertheless, results in whole-corn mini silos indicated that, although acid detergent fiber (ADF) was significantly reduced by this strain (3% compared with the uninoculated (UN) group), L. johnsonii ETC150 and CRL1446 not only induced similar ADF reduction but also reduced dry matter (DM) loss (by 7.3% and 6.5%, respectively) compared with the UN group. Additionally, CRL1446 increased in vitro DM degradability by 10%. All treatments reduced gas losses when compared with the UN group. The potential probiotic features of these strains, as well as their beneficial impact on corn fermentation shown in this study, encourage further studies as enhancers in animal production.

Rapid identification of new isolates of Acidipropionibacterium acidipropionici by fluorescence in situ hybridization (FISH).

Acidipropionibacterium acidipropionici is widely used for many applications, such as propionic acid production, cereal silage, and also as probiotic. Due to this plethora of applications, new isolates of A. acidipropionici with improved features are being searched for. These new isolates must be accurately identified, however, most approaches become expensive and time-consuming when the number of isolates is high. On the contrary, fluorescence in situ hybridization allows the affordable, reliable, and rapid identification of microorganisms in pure cultures and environmental and medical samples. Therefore, the aim of this work was to apply a fluorescent in situ hybridization probe for the reliable identification of new A. acidipropionici isolates. To this end, probe Pap446, specific for A. acidipropionici, was validated by hybridization assays with strains of this species from different origins, other species of the same genus or family, and unrelated genera. Eight isolates with propionibacterium characteristics were obtained from milk and feces of cows. Probe Pap446, hybridized only with isolates III and VI. The identity of these isolates was further confirmed by PCR using group and species-specific primers for propionibacteria and 16S rDNA sequencing.

Protection of the intestinal epithelium of poultry against deleterious effects of dietary lectins by a multi-strain bacterial supplement.

The intake of antinutritional factors produce impairment on the intestinal digestive function, impeding the efficient use of nutrients. Probiotics could be useful in poultry breeding to prevent negative effects of antinutritional factors, like the dietary lectins soybean agglutinin (SBA) and wheat germ agglutinin (WGA). Therefore, this investigation aimed to verify that SBA and wheat, which contains WGA, exert harmful effects on the intestinal mucosa and the digestive system of young poultry, and determine if the administration of probiotics able to capture lectins could counteract their effects. The trials performed demonstrated that a mixture of Bifidobacterium infantis CRL 1395, Enterococcus faecium LET 301, Lactobacillus salivarius LET 201, L. reuteri LET 210, and Propionibacterium acidipropionici LET 103, strains with ex vivo ability to interfere with the interaction of lectins and epithelial cells, has no negative effect on young chickens health. Middle levels of SBA, as well as wheat as a source of WGA, resulted in lower activities of intestinal and brush border enzymes and alterations in the integrity and morphological parameters of the chicks jejunal mucosa. The bacteria blend increased the activity of several digestive enzymes and the intestinal maturation marker alkaline phosphatase in birds fed with a conventional diet. Besides, it partially countered the deleterious effects of increased content of SBA, as well as the negative effect of a dietary source of WGA, on digestive enzymes activity and intestinal mucosa integrity. The results highlight the capability of multifunctional bacterial mixtures to protect the digestive system of avian against residual dietary lectins.

Dairy propionibacteria prevent the proliferative effect of plant lectins on SW480 cells and protect the metabolic activity of the intestinal microbiota in vitro.

Plant lectins are specific carbohydrate-binding proteins that are widespread in legumes such as beans and pulses, seeds, cereals, and many plants used as farm feeds. They are highly resistant to cooking and digestion, reaching the intestinal lumen and/or blood circulation with biological activity. Since many legume lectins trigger harmful local and systemic reactions after their binding to the mucosal surface, these molecules are generally considered anti-nutritive and/or toxic substances. In the gut, specific cell receptors and bacteria may interact with these dietary components, leading to changes in intestinal physiology. It has been proposed that probiotic microorganisms with suitable surface glycosidic moieties could bind to dietary lectins, favoring their elimination from the intestinal lumen or inhibiting their interaction with epithelial cells. In this work, we assessed in vitro the effects of two representative plant lectins, concanavalin A (Con A) and jacalin (AIL) on the proliferation of SW480 colonic adenocarcinoma cells and metabolic activity of colonic microbiota in the absence or presence of Propionibacterium acidipropionici CRL 1198. Both lectins induced proliferation of colonic cells in a dose-dependent manner, whereas ConA inhibited fermentative activities of colonic microbiota. Pre-incubation of propionibacteria with lectins prevented these effects, which could be ascribed to the binding of lectins by bacterial cells since P. acidipropionici CRL 1198 was unable to metabolize these proteins, and its adhesion to colonic cells was reduced after reaction with Con A or AIL. The results suggest that consumption of propionibacteria at the same time as lectins could reduce the incidence of lectin-induced alterations in the gut and may be a tool to protect intestinal physiology.

Ocurrencia de Salmonella en huevos destinados a consumo humano en la provincia de Tucumán / Occurrence of Salmonella in eggs intended for human consumption in the province of Tucumán

INTRODUCCIÓN La presencia en huevos de Salmonella enterica serovariedad Enteritidis, Salmonella enterica serovariedad Thyphimurium y bacterias Gram-negativas potencialmente patógenas, constituyen una preocupación a nivel mundial debido a su gran impacto en salud pública ya que pueden ingresar a la cadena alimentaria afectando a los consumidores. OBJETIVO Establecer la presencia S. Enteritidis, S. Thyphimurium y otras bacterias Gram-negativas potencialmente patógenas en huevos para consumo humano provenientes de granjas de gallinas ponedoras y establecimientos destinados a su comercialización en la provincia de Tucumán y estudiar sus perfiles de sensibilidad/resistencia frente a antibióticos empleados en medicina humana y medicina veterinaria. MÉTODOS Se propuso un estudio de tipo descriptivo-exploratorio. La población objeto fueron huevos adquiridos en comercios de la capital de la provincia y en granjas de gallinas ponedoras de los departamentos de Burruyacú, Simoca y Trancas. Se realizó la toma de muestras para la determinación cualitativa de S. Enteritidis, S. Thyphimurium y bacterias Gram-negativas potencialmente patógenas y se llevaron a cabo encuestas sobre Buenas Prácticas de Manufacturas (BPM) a fin de evaluar su aplicación en las granjas bajo estudio. RESULTADOS No se detectó la presencia de Salmonella sp. en ninguna de las muestras obtenidas en la capital (n=360) o en las granjas de gallinas ponedoras (n=300). Sin embargo, se determinó contaminación de las cáscaras de huevos con diferentes enterobacterias siendo E. coli la microflora predominante, la cual mostró resistencia a diferentes antibióticos. Se determinó la aplicación de BPM básicas en la mayoría de las granjas estudiadas. DISCUSIÓN Los datos revelan la presencia en la superficie de huevos de bacterias potencialmente patógenas resistentes a antibióticos de uso común, aún en ausencia de señales visibles de contaminación (heces). Por lo que resulta importante promover estrategias de prevención a lo largo de la cadena de producción y distribución del productos

Cytotoxic damage of soybean agglutinin on intestinal epithelial cells of broiler chicks: in vitro protection by Bifidobacterium infantis CRL1395.

Plant lectins, which are proteins/glycoproteins present in a wide range of vegetables, fruits, cereals and beans, are resistant to digestive enzymes and food cooking temperatures. They bind reversibly to specific glycosidic residues expressed on the membrane of intestinal epithelial cells (IEC) and cause anti-nutritional effects in humans and animals. Soybean lectin (SBA) has been detected in poultry diets, and its ability to bind to the intestinal epithelium has been reported. The development of new methods for removing SBA from feeds or to prevent interaction with the intestinal mucosa is of interest. In this study, the in vitro cytotoxicity of SBA on IEC of chicks was demonstrated for the first time. The LD50, assessed after 2 h exposure of IEC to SBA, was 6.13 µg mL(-1) The ability of Bifidobacterium infantis CRL1395 to bind SBA on the bacterial envelope was confirmed, and prevention of IEC cytotoxicity by lectin removal was demonstrated. Safety of B. infantis CRL1395, resistance to gastrointestinal stress and adhesion were also determined. It was concluded that the early administration of B. infantis CRL1395 to chicks would effectively reduce the toxicity of SBA. Besides, it would favour the colonization of the gut with a beneficial microbiota.

Selection of indigenous lactic acid bacteria to reinforce the intestinal microbiota of newly hatched chicken: relevance of in vitro and ex vivo methods for strains characterization.

Based on the natural benefits of the indigenous microbiota, lactic acid bacteria (LAB) from poultry origin were isolated from hens and broilers intestine, and their probiotic potential was further studied. The tolerance to digestion, adhesion, capture of a mannose-binding lectin, absence of virulent factors and antibiotic resistances were studied. Different in vitro and ex vivo assays were performed to select tolerant and adherent strains because standardized protocols have not been defined. Fourteen strains highly tolerant to gastrointestinal digestion were genetically identified. Hydrophobic surfaces were not required for the bacterial adhesion and only nine strains adhered ex vivo to the intestinal mucosa. Three strains captured a lectin of the same specificity of Type-1 fimbriae. Virulence factors were absent but some strains evidenced multiple antibiotic resistances. These results provide bases for a future standardization of methods for the selection of probiotic strains intended to reinforce the microbiota of newly hatched chickens.

Physiological and functional characteristics of Propionibacterium strains of the poultry microbiota and relevance for the development of probiotic products.

The prevention and control of pathogens colonization through probiotics administration in poultry feeding is of increasing interest. The genus Propionibacterium is an attractive candidate for the development of probiotic cultures as they produce short chain fatty acids (SCFA) by carbohydrates fermentation. The presence of strains of this genus in hens of conventional production systems and backyard hens was investigated. Propionibacteria were isolated from the intestine and identified by physiological and biochemical tests. PCR amplification of the 16S rRNA gene of the isolates was performed and products were compared with sequences from databases. The presence of the genus Propionibacterium was demonstrated in 26% of hens and Propionibacterium acidipropionici and Propionibacterium avidum were the identified species. A comparative study of their physiological and functional characteristics was performed. P. acidipropionici strains were the most resistant to in vitro gastrointestinal digestion, but the adhesion to intestinal tissue was strain dependent. Some differences were found between both species with respect to their growth and SCFA production in an in vitro cecal water model, but all the strains were metabolically active. The production of SCFA in cecal slurries inoculated with the strain P. acidipropionici LET 105 was 30% higher than in non-inoculated samples. SCFA concentrations obtained were high enough to inhibit Salmonella enterica serovar Enteritidis when assayed in a cecal water model. P. acidipropionici LET 105 was also able to compete with Salmonella for adhesion sites on the intestinal mucosa in ex vivo assays. Results contribute to the knowledge of the species diversity of the genus Propionibacterium in the intestine of poultry and provide evidence of their potential for probiotics products development.

Effect of functional buffalo cheese on fatty acid profile and oxidative status of liver and intestine of mice.

The objective of this study was to determine the influence of administration of buffalo dairy products on lipid content and conjugated linoleic acid (CLA) incorporation on liver and intestine of mice. Buffalo cheeses were selected according to nutritional properties and CLA content. Cheeses were previously manufactured using as adjunct culture bacteria with probiotic or technological properties. BALB/c mice were fed for 28 days, and then a single dose of 1,2-dimethylhydrazine (DMH) as oxidant agent was administered before the influence of diet and DMH on antioxidant status in tissues was evaluated. Mice fed buffalo cheese showed the highest body weight gain (P < .05). Polyunsaturated fatty acid (PUFA) content in foods was very different, but total PUFA incorporation was similar in mouse tissues. CLA was only detected in fat tissues of mice fed dairy products, with cis-9, trans-11 being the major isomer. A higher linolenic (C(18:3)) acid content was found in tissues of mice fed commercial diet (control group), and it was partially replaced by CLA in groups receiving buffalo milk or cheese. Lipoperoxides (thiobarbituric acid-reactive substances) were higher in tissues of the control group with or without DMH administration, and DMH had a cytotoxic effect on colon cells (P < .05). Superoxide dismutase (SOD) and catalase activities in liver and intestine were similar among animals, with a slight increase of SOD detected after DMH treatment. Consumption of buffalo dairy products did not affect the oxidative status of mice tissues even after DMH application. In the present study, a protective effect of buffalo cheese and milk on intestine cells was determined.

Fluorescence in situ hybridization for detection of classical propionibacteria with specific 16S rRNA-targeted probes and its application to enumeration in Gruyère cheese.

The classical or dairy propionibacteria have well-documented industrial applications and have been proposed for probiotic applications. Given their industrial importance it is necessary to employ fast and reliable techniques to monitor the growth during products elaboration, industrial fermentations or the intestinal transit. Therefore, the aim of this investigation was to design oligonucleotide probes targeting the 16S rRNA of dairy propionibacteria and optimise the fluorescence in situ hybridization (FISH) protocol to detect these bacteria. Two specific probes were in silico designed to detect Propionibacterium freudenreichii and P. jensenii, named Pfr435 and Pj446 respectively. The FISH protocol was optimised for the hybridisation of propionibacteria cells with the universal probe Eub338 and the designed probes. These probes were assayed in situ for their specificity to hybridise species of propionibacteria by observation using fluorescence microscopy and results were compared with the probe Pap446 previously designed for P. acidipropionici. Probes Pap446, Pfr435 and Pj446 were also evaluated by fluorescence spectrophotometry to assess the influence of cells physiological state during growth in batch culture in the fluorescence intensity. The maximum fluorescence intensity was observed at the onset of the stationary phase of growth and was then reduced. However, changes on the cells permeability did not reduce the efficiency of 16S rRNA hybridisation with the fluorescence-labelled probes. Propionibacteria counts obtained by FISH and plate count methods were compared in a commercial Gruyère cheese. The results showed that this method can be used as a rapid technique for the enumeration of these bacteria in cheese samples.

Some factors affecting the adherence of probiotic Propionibacterium acidipropionici CRL 1198 to intestinal epithelial cells.

Adhesion to the intestinal mucosa is generally considered an important property of probiotic microorganisms and has been related to many of their health benefits. This study investigated some factors that could affect or be involved in the adherence of Propionibacterium acidipropionici CRL 1198, a dairy strain with suggested probiotic effects and high adherence in vitro and in vivo to intestinal epithelial cells. In vitro adhesion of propionibacteria was decreased by gastric digestion but not affected by bile and pancreatic enzymes. Adherence was also decreased by pretreatment of bacterial cells with protease, sodium metaperiodate, and trichloroacetic acid, revealing that different features of the cell surface, like protein factors, carbohydrates, and teichoic acids, are involved in the process. Adherence to intestinal epithelial cells was enhanced by calcium and was dependent on other divalent cations. Adhesion to intestinal mucus was also demonstrated. The results should explain the metabolic effects in the host previously obtained with this strain and support the potential of Propionibacterium for development of new probiotics.

Identification of lactic acid bacterial and enterobacteria: a technique in microplates

En este trabajo se desarrolla una microtécnica para la identificación de bacterias lácticas y bacterias enteropatogénicas. Los resultados fueron comparados con los obtenidos por el método convencional. se logró una reducción del volumen final y del tiempo de incubación para el estudio de fermentación de azúcares y otras propiedades bioquímicas en comparación con el ensayo tradicional. La correlación obtenida entre el ensayo en microplaca y el método convencional fue superior al 90 por ciento. La preparación de las microplacas con los diferentes reactivos pueden ser conservadas bajo condiciones de refrigeración durante 30 días antes de su empleo. La microtécnica puede ser considerada como un método sencillo, rápido y económico