Molecular Monitoring of the Fecal Microbiota of Healthy Human Subjects during Administration of Lactulose and Saccharomyces boulardii

Diet is a major factor in maintaining a healthy human gastrointestinal tract, and this has triggered the development of functional foods containing a probiotic and/or prebiotic component intended to improve the host's health via modulation of the intestinal microbiota. In this study, a long-term placebo-controlled crossover feeding study in which each subject received several treatments was performed to monitor the effect of a prebiotic substrate (i.e., lactulose), a probiotic organism (i.e., Saccharomyces boulardii), and their synbiotic combination on the fecal microbiota of three groups of 10 healthy human subjects differing in prebiotic dose and/or intake of placebo versus synbiotic. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was used to detect possible changes in the overall bacterial composition using the universal V₃ primer and to detect possible changes at the subpopulation level using group-specific primers targeting the Bacteroides fragilis subgroup, the genus Bifidobacterium, the Clostridium lituseburense group (cluster XI), and the Clostridium coccoides-Eubacterium rectale group (cluster XIVa). Although these populations remained fairly stable based on DGGE profiling, one pronounced change was observed in the universal fingerprint profiles after lactulose ingestion. Band position analysis and band sequencing revealed that a band appearing or intensifying following lactulose administration could be assigned to the species Bifidobacterium adolescentis. Subsequent analysis with real-time PCR (RT-PCR) indicated a statistically significant increase (P < 0.05) in total bifidobacteria in one of the three subject groups after lactulose administration, whereas a similar but nonsignificant trend was observed in the other two groups. Combined RT-PCR results from two subject groups indicated a borderline significant increase (P = 0.074) of B. adolescentis following lactulose intake. The probiotic yeast S. boulardii did not display any detectable universal changes in the DGGE profiles, nor did it influence the bifidobacterial levels. This study highlighted the capacity of an integrated approach consisting of DGGE analysis and RT-PCR to monitor and quantify pronounced changes in the fecal microbiota of healthy subjects upon functional food administration.     

Effect of prebiotic substances on growth,fatty acid profile and probiotic characteristics of <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> NM101-1

Utilization of both probiotics and prebiotics in diet supplements and food products has gained a great interest because of their health benefits. In the present study, the effect of 6 commercially available prebiotic substances on the growth, acidifying activity, fatty acid profile and probiotic characteristics of Lactobacillus brevis NM101-1 was investigated in vitro for the development of synbiotic preparations. The results indicated the selective fermentability of prebiotics by the probiotic bacterial strain and absence of metabolism by pathogenic bacteria. Garlic and onion extracts as well as chicory flour as sources of inulin were the best carbon sources for growth and acidifying activity of the strain. The addition of onion extract to the medium exerted a significant influence on acetic acid production. However, the highest biosynthesis of lactic acid was recorded in the presence of glucose. Supplementation of MRS medium with prebiotic substances caused an increase in the ratio of unsaturated to saturated fatty acids of bacterial cells. Furthermore, resistance to gastrointestinal conditions, hydrophobicity and inhibition of bacterial pathogens as international guidelines for probiotics were enhanced by a combination of probiotic L. brevis and prebiotics which indicated that a convenient prebiotic substance have to be chosen for each probiotic bacterial strain for potential synbiotic preparation.     

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms'' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.     

Characterization and Antioxidant Property of Probiotic and Synbiotic Yogurts

The effect of a prebiotic (fructooligosaccharides) or a synbiotic components (prebiotic and probiotic) on the viability, proteolysis and antioxidant properties of probiotic and synbiotic yogurt during 28?days of storage at 4?°C has been investigated. Yogurt starters in conjunction with either probiotic bacteria Lactobacillus plantarum CFR 2194, Lactobacillus fermentum CFR 2192 and/or fructooligosaccharides (FOS) were used for yogurt preparation. Titratable acidity and pH of all yogurt samples followed a similar pattern of increase or decrease during storage. Proteolysis in synbiotic yogurts was found to be significantly (P?<?0.05) higher in comparison with that of control. The addition of prebiotics had no effect (P?=?0.17888) on the viability of yogurt starters during cold storage. No observable changes in the viability of probiotic cultures in probiotic groups. However, supplementation of FOS affected the growth significantly (P?<?0.05) in promoting the growth of L. plantarum and L. fermentum. Antioxidant activities, the index of nutritional value of yogurt, were monitored. Results showed that the DPPH-radical-scavenging activity (85?%) in synbiotic yogurt containing L. plantarum and FOS was significantly higher (P?<?0.05) in comparison with that of control yogurt (72?%). Total phenolics and the ferric reducing power were highest in synbiotic yogurts in comparison with that of other test samples during the entire period of storage. Addition of selected probiotics with FOS thus resulted in an improved functionality of yogurt.     

In vitro screening of probiotic lactic acid bacteria and prebiotic glucooligosaccharides to select effective synbiotics

Probiotics and prebiotics have been demonstrated to positively modulate the intestinal microflora and could promote host health. Although some studies have been performed on combinations of probiotics and prebiotics, constituting synbiotics, results on the synergistic effects tend to be discordant in the published works. The first aim of our study was to screen some lactic acid bacteria on the basis of probiotic characteristics (resistance to intestinal conditions, inhibition of pathogenic strains). Bifidobacterium was the most resistant genus whereas Lactobacillus farciminis was strongly inhibited. The inhibitory effect on pathogen growth was strain dependent but lactobacilli were the most effective, especially L. farciminis. The second aim of the work was to select glucooligosaccharides for their ability to support the growth of the probiotics tested. We demonstrated the selective fermentability of oligodextran and oligoalternan by probiotic bacteria, especially the bifidobacteria, for shorter degrees of polymerisation and absence of metabolism by pathogenic bacteria. Thus, the observed characteristics confer potential prebiotic properties on these glucooligosaccharides, to be further confirmed in vivo, and suggest some possible applications in synbiotic combinations with the selected probiotics. Furthermore, the distinctive patterns of the different genera suggest a combination of lactobacilli and bifidobacteria with complementary probiotic effects in addition to the prebiotic ones. These associations should be further evaluated for their synbiotic effects through in vitro and in vivo models.     

Molecular monitoring of the fecal microbiota of healthy human subjects during administration of lactulose and Saccharomyces boulardii

Diet is a major factor in maintaining a healthy human gastrointestinal tract, and this has triggered the development of functional foods containing a probiotic and/or prebiotic component intended to improve the host's health via modulation of the intestinal microbiota. In this study, a long-term placebo-controlled crossover feeding study in which each subject received several treatments was performed to monitor the effect of a prebiotic substrate (i.e., lactulose), a probiotic organism (i.e., Saccharomyces boulardii), and their synbiotic combination on the fecal microbiota of three groups of 10 healthy human subjects differing in prebiotic dose and/or intake of placebo versus synbiotic. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons was used to detect possible changes in the overall bacterial composition using the universal V(3) primer and to detect possible changes at the subpopulation level using group-specific primers targeting the Bacteroides fragilis subgroup, the genus Bifidobacterium, the Clostridium lituseburense group (cluster XI), and the Clostridium coccoides-Eubacterium rectale group (cluster XIVa). Although these populations remained fairly stable based on DGGE profiling, one pronounced change was observed in the universal fingerprint profiles after lactulose ingestion. Band position analysis and band sequencing revealed that a band appearing or intensifying following lactulose administration could be assigned to the species Bifidobacterium adolescentis. Subsequent analysis with real-time PCR (RT-PCR) indicated a statistically significant increase (P < 0.05) in total bifidobacteria in one of the three subject groups after lactulose administration, whereas a similar but nonsignificant trend was observed in the other two groups. Combined RT-PCR results from two subject groups indicated a borderline significant increase (P = 0.074) of B. adolescentis following lactulose intake. The probiotic yeast S. boulardii did not display any detectable universal changes in the DGGE profiles, nor did it influence the bifidobacterial levels. This study highlighted the capacity of an integrated approach consisting of DGGE analysis and RT-PCR to monitor and quantify pronounced changes in the fecal microbiota of healthy subjects upon functional food administration.     

Immunomodulatory function and in vivo properties of Pediococcus pentosaceus OZF, a promising probiotic strain

Some of the important properties of probiotics are the ability to survive during gastrointestinal transit and to modulate the immune functions. The objectives of the reported study were to assess in vivo gastrointestinal survival of orally administered Pediococcus pentosaceus OZF using an animal model BALB/c mice, and to examine its effects on the immune response. Following oral administration to mice, the ability of Pediococcus pentosaceus OZF to pass and survive through the mouse gastrointestinal system was investigated by analyzing the recovery of the strain in fecal samples. Microbiological and polymerase chain reaction (PCR) methods proved that the strain OZF could overcome specific conditions in the gastrointestinal tract of mice and reach the intestine alive after ingestion. To observe the effect of oral administration on immune response, IL-6, IL-12 and IFN-γ were measured by ELISA, and the strain OZF was found to cause increases in IL-6 synthesis in regularly fed mice. However, stimulation was carried out with various concentrations of bacterial ssDNA and heat killed cells of Pediococcus pentosaceus OZF. The heat killed cells of the strain OZF were shown to produce IFN-γ independently from IL-12. On the other hand, a significant difference between control and experimental group was noticed when lipopolysaccharide, a TLR4 (toll like receptor) ligand, was used. Overall, Pediococcus pentosaceus OZF may be a valuable probiotic strain for therapeutic uses. Nevertheless, further studies on the mechanisms of immunomodulatory effect will allow for better clarification of the immune functions of this strain.     

The Effect of Synbiotic Supplementation on Growth Parameters in Mild to Moderate FTT Children Aged 2–5 Years

Synbiotic (probiotic bacteria and prebiotic) has beneficial effects on the gastrointestinal tract. This study was designed to investigate the effect of synbiotic supplementation on the growth of mild to moderate failure to thrive (FTT) children. A randomized, triple-blind, placebo-controlled trial was conducted involving 80 children aged 2–5 years with mild to moderate FTT, who were assigned at random to receive synbiotic supplementation (10⁹ colony-forming units) or placebo for 30 days. The weights, height, and BMI were recorded in a structured diary, and the questionnaires were completed to monitor the numbers of infection episodes, gastrointestinal problems, admission to hospital, and appetite improvement during the study. Sixty-nine children completed the study. There were no differences in the demographic characteristic between the two groups. The mean weight was similar at baseline. After 30 days of intervention, the mean weight of the participants in the synbiotic group increased significantly than those in the placebo group (600?±?37 vs. 74?±?32 g/month P 0.000). BMI changes in synbiotic and placebo group were 0.44 and 0.07 kg/m², and that the differences among the two groups were significant.(P 0.045) Furthermore, the height increment in synbiotic and placebo group was 0.41 and 0.37 cm respectively with no significant difference (P 0.761). Administration of 30-day synbiotic supplementation may significantly improve weight and BMI in Iranian children with mild to moderate FTT, but there is no effect on the height in this study. Further studies should be designed to found out the effect of synbiotic on growth parameters in undernourished and well-nourished children.

     

A novel multi‐strain probiotic and synbiotic supplement for prevention of Clostridium difficile infection in a murine model

The protective effect of a multi‐strain probiotic and synbiotic formulation was evaluated in C57BL/6 mice infected with Clostridium difficile (CD) NAP1/027. Antibiotic‐treated mice were divided into the following four groups: Group 1, fed with a synbiotic formulation consisting of Lactobacillus plantarum F44, L. paracasei F8, Bifidobacterium breve 46, B. lactis 8:8, galacto‐oligosaccharides, isomalto‐oligosaccharides, and resistant starch; Group 2, fed with the same four probiotic strains as Group 1; Group 3, fed with the same prebiotic supplements as Group 1 for 7 days before CD infection; and Group 4 (control group) antibiotic treated and infected with NAP1/027 strain. Feces and cecal contents were collected for microbial cell viability, quantitative PCR (qPCR), toxin analyses and histopathology. Synbiotics‐ and probiotics‐fed mice showed a significant increase in total bifidobacteria (P < 0.05). The total lactobacilli count was increased in Group 1. Tests for cecal toxins were negative in Group 2 mice, whereas one sample each from Group 1 and 3 was positive. qPCR of cecal contents showed significant reduction in NAP1/027 DNA copies in Groups 1 and 2 and significantly higher numbers of B. breve 46, L. plantarum F44, and L. paracasei F8 in Groups 1 and 2 (P < 0.05); these changes were much less pronounced in Groups 3 and 4. Our findings indicate that the newly developed synbiotic or multi‐strain probiotic formulation confers protection against NAP1/027 infection in C57BL/6 mice. This holds promise for performing human studies.     

Identification of plant-associated enterococci

AIMS: To employ an in vitro screening regime to select a probiotic Bifidobacterium strain to complement resistant starch (Hi-maizetrade mark) in a synbiotic yoghurt. METHODS AND RESULTS: Of 40 Bifidobacterium isolates examined, only B. lactis Laftitrade mark B94 possessed all of the required characteristics. This isolate hydrolysed Hi-maizetrade mark, survived well in conditions simulating passage through the gastrointestinal tract and possessed technological properties suitable for yoghurt manufacture. It grew well at temperatures up to 45 degrees C, and grew to a high cell yield in an industrial growth medium. In addition to resistant starch, the organism was able to utilize a range of prebiotics including inulin, and fructo-, galacto-, soybean- and xylo-oligosaccharides. Pulse field gel electrophoresis of restriction enzyme cut chromosomal DNA revealed that B. lactis Laftitrade mark B94 was very closely related to the B. lactis Type Strain (DSM 10140), and to the commercial strains B. lactis Bb-12 and B. lactis DS 920. However, B. lactis Laftitrade mark B94 was the only one of these isolates that could hydrolyse Hi-maizetrade mark. This phenotypic difference did not appear to be due to the presence of plasmid encoded amylase. Bifidobacterium lactis Laftitrade mark B94 survived without substantial loss of viability in synbiotic yoghurt containing Hi-maizetrade mark during storage at 4 degrees C for six weeks. CONCLUSION: Bifidobacterium lactis Laftitrade mark B94 is a promising new yoghurt culture that warrants further investigation to assess its probiotic potential. SIGNIFICANCE AND IMPACT OF THE STUDY: In vitro screening procedures can be used to integrate complementary probiotic and prebiotic ingredients for new synbiotic functional food products.     

Quantitative Real-Time PCR Assays To Identify and Quantify Fecal Bifidobacterium Species in Infants Receiving a Prebiotic Infant Formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5′ nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5′ nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% ± 9.8% to 73.4% ± 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% ± 1.92% and 8.11% ± 4.12%, respectively, versus 0.15% ± 0.11% and 1.38% ± 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Glycoprofiling Bifidobacterial Consumption of Galacto-Oligosaccharides by Mass Spectrometry Reveals Strain-Specific,Preferential Consumption of Glycans

Galacto-oligosaccharides (GOS) are versatile food ingredients that possess prebiotic properties. However, at present there is a lack of precise analytical methods to demonstrate specific GOS consumption by bifidobacteria. To better understand the role of GOS as prebiotics, purified GOS (pGOS) without disaccharides and monosaccharides was prepared and used in bacterial fermentation experiments. Growth curves showed that all bifidobacteria assayed utilized and grew on pGOS preparations. We used a novel mass spectrometry approach involving matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) to determine the composition of oligosaccharides in GOS syrup preparations. MALDI-FTICR analysis of spent fermentation media demonstrated that there was preferential consumption of selected pGOS species by different bifidobacteria. The approach described here demonstrates that MALDI-FTICR is a rapid-throughput tool for comprehensive profiling of oligosaccharides in GOS mixtures. In addition, the selective consumption of certain GOS species by different bifidobacteria suggests a means for targeting prebiotics to enrich select bifidobacterial species.Galacto-oligosaccharides (GOS) are nondigestible carbohydrates and versatile food ingredients that possess prebiotic properties (1). In addition, other health benefits have been reported to result from consumption of these oligosaccharides, such as stimulation of intestinal mobility and mineral absorption, elimination of ammonium, and colon cancer prevention, as well as protection against certain pathogenic bacterial infections (6, 11, 19).The physicochemical characteristics of GOS have enabled them to be incorporated in food as prebiotic ingredients. GOS have been of interest in acidic beverages and fermented milk formulations since they exhibit increased thermal stability in acidic environments compared to fructo-oligosaccharides (16, 21). Thus, in the past decade, the applications of GOS in human food products have included dairy products, sugar replacements, diet supplements, and infant formula (11).Commercial GOS preparations are produced by enzymatic treatment of lactose with β-galactosidases from different sources, such as fungi, yeast, or bacteria, which results in a mixture of oligomers with various chain lengths (1). Thus, the basic structure of GOS includes a lactose core at the reducing end, which is typically elongated with up to six galactose residues. Structural diversity in GOS preparations is dependent on the enzyme used in the transgalactosylation reaction and the experimental conditions used, such as pH and temperature (5).Considerable effort has been made to understand the effects of GOS in vivo, and most studies have described the impact of GOS on intestinal bacterial population shifts and production of short-chain fatty acids attributed to bacterial fermentation. While there have a been a variety of in vitro studies characterizing the growth of different gut microbes on GOS, the majority of these studies used commercially available preparations of GOS. These commercial preparations contain high concentrations of monosaccharides (i.e., galactose and glucose) and the disaccharide lactose, both of which remain in the product after the transgalactosylation reaction. However, monosaccharides are the preferred substrates for most microorganisms when they are available in a mixed-carbon source (2). Thus, to evaluate growth on GOS, removal of monosaccharides and lactose is helpful (15).An analytical method currently used to measure GOS in food and feed products is high-pH anion-exchange chromatography (HPAEC) coupled to analysis with a pulse amperometric detector (PAD) (4). Van Laere and coworkers have used this method to monitor GOS fermentation in Bifidobacterium adolescentis cultures (20). However, HPAEC-PAD analysis is time-consuming and thus a low-throughput method. More importantly, due to the detector, in HPAEC-PAD analysis there is a differential response to oligosaccharides with higher degrees of polymerization (DP). Thus, new analytical approaches are needed to specifically characterize the consumption of GOS and other prebiotics by probiotic bacteria.We have previously developed analytical methods employing high-mass-accuracy and high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) to characterize bacterial consumption of human milk oligosaccharides and fructo-oligosaccharides (9, 10, 14, 17). The matrix-assisted laser desorption ionization (MALDI)-FTICR method is a sensitive and robust analytical method with high-performance capability, and it allows rapid and unambiguous assignment of oligosaccharide signals.The aims of the present study were to investigate the oligosaccharide composition of GOS syrup preparations using MALDI-FTICR MS, to test lactose-free purified GOS (pGOS) as a sole carbon source in bifidobacterial fermentation experiments, and to determine the pGOS consumption profile by MALDI-FTICR MS. Four major bifidobacterial phylotypes, B. adolescentis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, and Bifidobacterium longum subsp. longum, were used, and our results demonstrate that there is differential consumption of individual GOS species by various bifidobacteria, which provides a conceptual basis for targeted enrichment of specific bifidobacterial strains using specific GOS fractions.     

Lactobacillus salivarius CTC2197 Prevents Salmonella enteritidis Colonization in Chickens

A rifampin-resistant Lactobacillus salivarius strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent Salmonella enteritidis C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with S. enteritidis C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of L. salivarius CTC2197 in the first day chicken feed revealed that a concentration of 10⁵ CFU g⁻¹ was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days, L. salivarius CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the L. salivarius CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.     

Barcoded pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans

Prebiotics are selectively fermented ingredients that allow specific changes in the gastrointestinal microbiota that confer health benefits to the host. However, the effects of prebiotics on the human gut microbiota are incomplete as most studies have relied on methods that fail to cover the breadth of the bacterial community. The goal of this research was to use high throughput multiplex community sequencing of 16S rDNA tags to gain a community wide perspective of the impact of prebiotic galactooligosaccharide (GOS) on the fecal microbiota of healthy human subjects. Fecal samples from eighteen healthy adults were previously obtained during a feeding trial in which each subject consumed a GOS-containing product for twelve weeks, with four increasing dosages (0, 2.5, 5, and 10 gram) of GOS. Multiplex sequencing of the 16S rDNA tags revealed that GOS induced significant compositional alterations in the fecal microbiota, principally by increasing the abundance of organisms within the Actinobacteria. Specifically, several distinct lineages of Bifidobacterium were enriched. Consumption of GOS led to five- to ten-fold increases in bifidobacteria in half of the subjects. Increases in Firmicutes were also observed, however, these changes were detectable in only a few individuals. The enrichment of bifidobacteria was generally at the expense of one group of bacteria, the Bacteroides. The responses to GOS and the magnitude of the response varied between individuals, were reversible, and were in accordance with dosage. The bifidobacteria were the only bacteria that were consistently and significantly enriched by GOS, although this substrate supported the growth of diverse colonic bacteria in mono-culture experiments. These results suggest that GOS can be used to enrich bifidobacteria in the human gastrointestinal tract with remarkable specificity, and that the bifidogenic properties of GOS that occur in vivo are caused by selective fermentation as well as by competitive interactions within the intestinal environment.     

Genotypic adaptations associated with prolonged persistence of Lactobacillus plantarum in the murine digestive tract

Probiotic bacteria harbor effector molecules that confer health benefits, but also adaptation factors that enable them to persist in the gastrointestinal tract of the consumer. To study these adaptation factors, an antibiotic-resistant derivative of the probiotic model organism Lactobacillus plantarum WCFS1 was repeatedly exposed to the mouse digestive tract by three consecutive rounds of (re)feeding of the longest persisting colonies. This exposure to the murine intestine allowed the isolation of intestine-adapted derivatives of the original strain that displayed prolonged digestive tract residence time. Re-sequencing of the genomes of these adapted derivatives revealed single nucleotide polymorphisms as well as a single nucleotide insertion in comparison with the genome of the original WCFS1 strain. Detailed in silico analysis of the identified genomic modifications pinpointed that alterations in the coding regions of genes encoding cell envelope associated functions and energy metabolism appeared to be beneficial for the gastrointestinal tract survival of L. plantarum WCFS1. This work demonstrates the feasibility of experimental evolution for the enhancement of the gastrointestinal residence time of probiotic strains, while full-genome re-sequencing of the adapted isolates provided clues towards the bacterial functions involved. Enhanced gastrointestinal residence is industrially relevant because it enhances the efficacy of the delivery of viable probiotics in situ.     

Growth promoter,immune response,and histopathological change of prebiotic,probiotic and synbiotic bacteria on Nile tilapia

This study aimed at determining the influence of prebiotic, probiotic, and synbiotic supplemented diets on Oreochromis niloticus. Fish with initial body weight (25.8 ± 1.2) g and length range from (13.5 ± 1.5) cm were collected and randomized to four dietary treatments for 60 days. Furthermore, fish were divided into three groups in triplicate; A0 control (-ve), A1 control (+ve) infected with V.anguillarium, and a third non-treated group. Moreover, the third group further separated into two groups, A and B. Group (A) was treated with prebiotic, probiotic, and symbiotic (A2, A3, and A4), while group (B) was infected with V.anguillarium then treated with prebiotic, probiotic and symbiotic (A5, A6, and A7). The results revealed that all treatments supplemented with synbiotics represented highly significant increase (p ≤ 0.05) in (SGR), BWG percentage, relative growth rate (%), lysozyme activity, IMG, SOD, and CAT. At the same time, they exhibited a significant decrease in MAD and FCR. Besides, fish that feed dietary supplementation with prebiotics, probiotics, and synbiotics revealed a significant increase in RBCs, WBCs, and Hb. In contrast, they showed a significant decrease in ALT, AST, albumin, total protein, globulin, creatinine, and urea compared with control. Additionally, high survival rates were recorded in groups that received a diet supplemented with probiotics, followed by prebiotics and synbiotics.     

Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074^T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.     

Effects of Simple and Microencapsulated <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> With or Without Inulin on the Broiler Meat Quality Infected by Avian Influenza Virus (H<Subscript>9</Subscript>N<Subscript>2</Subscript>)

The aims of the present study were to investigate the effects of simple and microencapsulated Lactobacillus acidophilus as probiotic with or without inulin as prebiotic on meat quality of broiler infected by avian influenza virus (H₉N₂). Two hundred-day-old chicks were randomly allocated into 14 groups based on simple, microencapsulated probiotic and prebiotic and based on vaccination and challenge with H₉N₂ virus. Groups 1–7 contained 20 chicks, and groups 8–12 and 14 contained 10 chicks. Group 13 was derived from group 1 with 10 chicks at challenge day with avian influenza virus (AIV). Half of the groups were vaccinated by H₉N₂ vaccine on day 5. All groups except the negative control and positive vaccine control were challenged with 10^6.5 EID₅₀ of low-virulence H₉N₂ AIV at day 21. Each bird was received 10⁹ CFU of simple or microencapsulated probiotic on days 0 and 17 by gavage. Prebiotic as dose as 0.1 % of feed weight was used daily. Increase in water-holding capacity, dry matter, ash and protein content, and decrease in dripping loss plus beneficial changes in lightness and redness of breast meat were detected in response to probiotic especially microencapsulated synbiotic. In conclusion, probiotic alone or with prebiotic was able to improve the physicochemical properties of chicken breast muscle in both healthy and AIV-infected chickens.