Silvia N. González

Adhesion of Dairy Propionibacteria to Intestinal Epithelial Tissue In Vitro and In Vivo

Adhesion to the intestinal mucosa is a desirable property for probiotic microorganisms and has been related to many of their health benefits. In the present study, 24 dairy Propionibacterium strains were assessed with regard to their hydrophobic characteristics and their autoaggregation and hemagglutination abilities, since these traits have been shown to be indicative of adherence in other microorganisms. Six strains were further tested for their capacity to adhere to ileal epithelial cells in vitro and in vivo. The results of the study showed that propionibacteria were highly hydrophilic, and hemagglutination and autoaggregation were properties not commonly found among these microorganisms. No relationship was found between surface characteristics and adhesion ability, since hemagglutinating, autoaggregating, and nonautoaggregating bacteria were able to adhere to intestinal cells both in vitro and in vivo. Microscopic examination revealed that autoaggregating cells adhered in clusters, with adhesion being mediated by only a few bacteria, whereas the hemagglutinating and nonautoaggregating strains adhered individually or in small groups making contact with each epithelial cell with the entire bacterial surface. The in vitro assessment of adhesion was a good indication of the in vivo association of propionibacteria with the intestinal epithelium. Therefore, the in vitro method presented here should be valuable in screening routinely adhesive properties of propionibacteria for probiotic purposes. The adhesion ability of dairy propionibacteria would prolong their maintenance in the gut and increase the duration of their provision of beneficial effects in the host, supporting the potential of Propionibacterium in the development of new probiotic products.

Biotherapeutic role of fermented milk.

Fermented milk was used as therapy in infantile diarrhoea due to post-gastroenteritis syndrome. This treatment eliminated the disease in 4.0 days (mean value, SD=2.8; n=13) and allowed patients to return to free feeding according to their age. The weight percentile variation during treatment with fermented milk (15 days) was higher in the patients showing 3rd degree malnutrition than in other children. Bacteriotherapy can restore faecal flora which has been lowered by diarrhoea. Our results showed that levels higher than 106 UFC lactobacilli/g of faeces correlated with a healthy status of the children. Clinical applications of fermented milk with a mixture ofLactobacillus casei andLactobacillus acidophilus in the prevention of gastrointestinal disorders are possible.

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.

Acetaldehyde Production by Strains Used as Probiotics in Fermented Milk

Lactic acid bacteria have diverse shunts for the metabolism of acetaldehyde, which is involved in the metabolism of carbohydrates, proteins and nucleic acids. In Lactobacillus acidophilus and Lactobacillus casei , strains isolated from feces of healthy children, acetaldehyde can be formed from different sources. Phosphotransacetilase, acetate kinase, aldehyde dehydrogenase and 2-deoxiriboaldolase activities were found in both strains. α-Carboxilase and threonine aldolase activities only occurred in L. acidophilus . In contrast, phosphoketolase activity was present in L. casei , but absent in the other strain studied. The accumulation of acetaldehyde in the growth medium is possible because the enzymes specific activities to form it are higher than those able to convert it to ethanol.

Fatty acid profile of pig meat after probiotic administration.

The aim of this work was to study the fatty acid profile of pig meat after probiotic administration. Thirty postweaned pigs (25 day old) were distributed into 2 groups: control (n = 15) and probiotic (n = 15). Each experimental group was fed ad libitum on a commercial diet for 35 days. Lactobacillus amylovorus and Enterococcus faecium mixed culture (10(8) CFU/ml)was daily orally delivered to the probiotic group. At the end of the assay, six pigs randomly selected from each group were slaughtered and muscle samples (Longissimus dorsi) were taken for fatty acid analysis. Tissues from the probiotic group animals exhibited an increase in monounsaturated and polyunsaturated fatty acids; furthermore, linoleic acid (C18:2), linolenic acid (18:3), and cis-9,trans-11 conjugated linoleic acid (CLA) concentrations were significantly higher (p < 0.05) compared to the control group. These results suggest probiotic administration could be useful to modify and improve the fatty acid profile of pig meat.

Prevention of Infections Produced by Escherichia coli and Listeria monocytogenes by Feeding Milk Fermented With Lactobacilli

Challenge studies were set up feeding Lactobacillus casei and Lactobacillus acidophilus fermented milk and two different pathogenic microorganisms: Listeria monocytogenes and enteroinvasive Escherichia coli . Mice were fed for 8 consecutive days with fermented milk and then challenged with the pathogens. The survival rate in control mice was 62% for Listeria and 83% for E. coli , while 100% protection was observed for the 20 d per vial in treated mice. Colonization of the liver and spleen by E. coli was markedly inhibited by pretreatment with fermented milk; the pathogen was not detected on the 5th day postchallenge. In the Listeria challenged mice, the pathogen was present in 1 to 2 log units lower than control up the 10th day. The levels of antipathogen sera and intestinal antibodies were 2 to 4 times higher in the treated mice, with lower values in the Listeria treated mice. The mechanism of protection in both types of infections was discussed. The results obtained suggested that milk fermented with L. casei and L. acidophilus could be used as a prophylactic against selected infections.

Study of adhesion of Lactobacillus casei CRL 431 to ileal intestinal cells of mice.

It is well known that the cell wall of Lactobacillus casei CRL 431, a strain present in probiotics, presents lectinlike surface molecules. Presence of these molecules stimulates the immune system. Given the role that lectins and lectinlike substances play in the adhesion phenomenon, it is probable that this is an initial stage in the immunostimulation produced by this bacterium. To confirm this, adhesion of this microorganism to exfoliated mouse ileal epithelial cells was studied in vitro. Other L. casei strains isolated from adult human intestines and one of dairy origin were also examined for their ability to adhere to ileal epithelial cells. Another strain, which was included in the present study, was Lactobacillus acidophilus CRL 730. L. casei strains isolated from humans showed good ability to adhere to ileal epithelial cells, whereas L. casei isolated from dairy origin did not. Adhesion was only observed at 37 degrees C and at a pH between 6 and 7.5. The exposure time needed for highest adhesion was 30 min. Presence of lectinlike substances on the surface of L. casei CRL 431 is important to this adhesion phenomenon, since adherence capacity was lost after removal of these substances.

Contribution of lactic acid bacteria esterases to the release of fatty acids in miniature ewe's milk cheese models.

The present work evaluates the contribution of esterase activities of lactic acid bacteria isolated from ewes dairy products to the release of free fatty acids (FFA) in ewes milk cheese models. At 60 days of ripening, single-strain cheeses Ov 409 and Ov 421 showed high levels of total FFA (3075 and 2494.62 mg/kg, respectively). Cheeses Ov 227-Ov 409 and Ov 421-Ov 409 presented high percentages of short-chain fatty acids (SCFA). The highest levels of volatile free fatty acids (VFFA) were detected in cheeses Ov 409, Ov 421-Ov 409, and Ov 421-Ov 227. Studies on esterase activities showed that these strains hydrolyzed alpha-naphthyl derivatives of fatty acids from C2 to C6, mainly associated with the wall-membrane fraction. The results showed that the strains studied contributed to the release of FFA during ripening of ewes milk cheese models. The increase of SCFA throughout ripening involves the action of esterases of starter strains.

Lactobacillus casei CRL 431 and Lactobacillus rhamnosus CRL 1224 as biological controls for Aspergillus flavus strains.

The effect of two species of lactobacilli, Lactobacillus casei CRL 431 and Lactobacillus rhamnosus CRL 1224, on growth of different Aspergillus flavus strains was determined. A. flavus strains (Ap, TR2, or CF80) were grown in LAPTg broth at 37 degrees C for 7 days as a single culture and in association with L. casei CRL 431 or L. rhamnosus CRL 1224 at initial inoculum ratios of 1:1, 1:10, and 1:100. In most cases, the mixed cultures had a lower fungal growth and a lower pH than the control cultures. Mycelial dry weight was reduced to 73 and 85% using L. casei CRL 431 and L. rhamnosus CRL 1224, respectively. The pH decrease in mixed cultures when the fungal mycelial dry weight is reduced may play an important role in inhibition. The number of viable bacteria was variably affected by fungal growth. These results indicate that L. casei CRL 431 and L. rhamnosus CRL 1224 may be useful as potential biocontrol agent against A. flavus.

Conjugated Linoleic and Linolenic Acid Production by Bacteria: Development of Functional Foods

© 2012 Van Nieuwenhove et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Conjugated Linoleic and Linolenic Acid Production by Bacteria: Development of Functional Foods