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.

Cane molasses and oligofructose in the diet of laying hens improves the mineral content of eggs and meat.

Poultry-based foods contribute to human health due to their high nutrient value. Previously, it was shown that short-chain fatty acids (SCFAs) produced by in vitro intestinal fermentation of a molasses and oligofructose mixture (M-O) stimulated iron and calcium transport through the colonic epithelium of laying hens. However, the real impact of including M-O mixture in the diet on the mineral content of poultry products had not yet been demonstrated. In this study, Hy-Line W-36 leghorn hens were assigned into two groups that either received a conventional diet or a diet supplemented with cane molasse and oligofructose, over a period of 42 days. The weight of the animals and their eggs, blood parameters and intestinal epithelium integrity were determined. Intestinal bacteria, their fermentation products, and the mineral content of eggs, bones and muscles were also assessed. The experimental diet proved to be safe, favored the proliferation of SCFA producing bacteria in the intestines, led to higher concentration of acids (mainly SCFA) in the digesta, and induced the elongation of microvilli at the apical tip of enterocytes. Mineral content of eggs and meat were improved after four weeks of feeding with the experimental diet compared to the conventional one. Higher iron content was observed in the edible portion of eggs and leg muscle, and higher calcium content was observed in the egg edible portion and shell in hens fed the supplemented diet. This feeding strategy could be useful to improve the mineral content of poultry products and therefore human nutrition, while diversifying molasses applications.

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.

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.

Properties of different lactic acid bacteria isolated from Apis mellifera L. bee-gut.

Eight strains belonging to Lactobacillus spp. and five to Enterococcus spp. were isolated from the gut of worker Apis mellifera L. bees. Studies based on 16S rRNA sequencing revealed that AJ5, IG9, A15 and CRL1647 strains had a 99% identity with Lactobacillus johnsonii, while SM21 showed a 99% similarity with Enterococcus faecium. L. johnsonii CRL1647, AJ5 and IG9 were high lactic acid producers (values were between 177 and 275 mM), and in vitro they inhibited different human food-borne pathogens and Paenibacillus larvae, the American foulbrood agent. This bacterium was the most sensitive to the lactic acid effect being inhibited by 44 mM of this metabolite. L. johnsonii CRL1647, AJ5 and IG9 also presented important surface properties. These cells showed between 77% and 93% of auto-aggregation. The preliminary study of the chemical nature of the aggregating factors revealed that the molecules involved in the surface of each L. johnsonii strain were quite complex; and something of a peptidic nature was mainly involved. E. faecium SM21 produced bacteriocin-like compounds with anti-Listeria effects. Furthermore, a band close to 6.0-7.5 kDA was detected by SDS-PAGE studies, and the entA, B and P structural genes were amplified by PCR reactions. For the first time, bee-gut associated L. johnsonii and E. faecium strains have been isolated, identified, cultivated and some of their functional properties reported.

Enterococcus faecium isolated from honey synthesized bacteriocin-like substances active against different Listeria monocytogenes strains.

Four Enterococcus faecium strains, isolated from honeycombs (C1 and M2d strains) and feral combs (Mori1 and M1b strains) secreted antimicrobial substances active against fourteen different Listeria spp. strains. The antimicrobial compound(s) present in the cell free supernatant were highly thermostable (121 degrees C for 15 min) and inactivated by proteolytic enzymes, but not by alpha-amylase and lipase, thus suggesting a peptidic nature. Since the structural bacteriocin gene determinants of enterocins A and B were PCR amplified from the four E. faecium isolates, only the bacteriocin produced by strain C1 was further characterized: it showed a broad band of approximately 4.0-7.0 kDa in SDS-PAGE and was bactericidal (4 log decrease) against L. monocytogenes 99/287. L. monocytogenes 99/287R, a clone spontaneously resistant to the enterocin produced by E. avium DSMZ17511 (ex PA1), was not inhibited by the enterocin-like compounds produced by strain C1. However, it was inhibited in mixed culture fermentations by E. faecium C1 and a bacteriostatic effect was observed. The bacteriocin-producer Enterococcus strains were not haemolytic; gelatinase negative and sensitive to vancomycin and other clinically relevant antibiotics.

Bacteriocin from honeybee beebread Enterococcus avium, active against Listeria monocytogenes.

Enterococcus avium isolated from Apis mellifera beebread produces a thermoresistant bacteriocin with a strain-dependent inhibitory effect on Listeria and without effect on gram-negative bacteria. The bacteriocin appeared to be a polypeptide of about 6 kDa. Genetic analyses revealed no extrachromosomal material in E. avium.

Probiotic bacteriotherapy in chronic infantile diarrhea

La administración de un producto lácteo conteniendo Lactobacillus casei y Lactobacillus acidophillus fue usada como terapia probiótica para diarrea crónica en 170 niños cuyas edades estaban comprendidas entre 4 meses y 4 años. El tiempo promdedio de duración de diarrea en estos niños, antes del tratamiento probiótico, era de 50 días. Los síntomas clínicos se eliminaron al cabo de 4 días de terapia probiótica y ninguno de los pacientes incurrió nuevamente en diarrea hasta el final del estudio. Los recuentos bacterianos en heces sugirieron que los lactobacilos probióticos habían normalizado la flora láctica intestinal. El producto lácteo fermentado ensayado en este trabajo puede ser considerado un alimento beneficioso

Inhibition of Enteropathogens by Lactobacilli Strains Used in Fermented Milk.

The inhibitory effect of lactobacilli on the growth of different pathogens was studied. The degree of inhibition was measured by disc assay in different media, showing that the inhibitory substance(s) can be extracellular and diffusible. The associative cultures were performed with lactobacilli and Escherichia coli in a relationship of 1:1 or 103:1. In the first experiments, the E. coli inhibition began at 9 h after culturing. In the experiments with lower levels of different intestinal pathogens, associative cultures were incubated for 4-6 h obtaining different degrees of inhibition with the highest being for E. coli and Klebsiella pneumoniae . After a day of incubation, we did not recover viable pathogens from the mixed cultures, while lactobacilli grew to 108-109 microorganisms per ml. The inhibition of pathogens was not due to pH alone, because the addition of different organic acids to culture media did not inhibit their normal growth. Lactobacillus casei and Lactobacillus acidophilus in this study can be considered a potential biotherapeutic treatment in patients infected with certain intestinal pathogens.