Yogurt, cultured fermented milk, and health: a systematic review.

Consumption of yogurt and other fermented products is associated with improved health outcomes. Although dairy consumption is included in most dietary guidelines, there have been few specific recommendations for yogurt and cultured dairy products. A qualitative systematic review was conducted to determine the effect of consumption of fermented milk products on gastrointestinal and cardiovascular health, cancer risk, weight management, diabetes and metabolic health, and bone density using PRISMA guidelines. English language papers in PubMed were searched, with no date restrictions. In total, 1057 abstracts were screened, of which 602 were excluded owing to lack of appropriate controls, potential biases, and experimental design issues. The remaining 455 papers were independently reviewed by both authors and 108 studies were included in the final review. The authors met regularly to concur, through consensus, on relevance, methods, findings, quality, and conclusions. The included studies were published between 1979 and 2017. From the 108 included studies, 76 reported a favorable outcome of fermented milks on health and 67 of these were considered to be positive or neutral quality according to the Academy of Nutrition and Dietetics' Quality Criteria Checklist. Of the 32 remaining studies, the study outcomes were either not significant (28) or unfavorable (4), and most studies (18) were of neutral quality. A causal relationship exists between lactose digestion and tolerance and yogurt consumption, and consistent associations exist between fermented milk consumption and reduced risk of breast and colorectal cancer and type 2 diabetes, improved weight maintenance, and improved cardiovascular, bone, and gastrointestinal health. Further, an association exists between prostate cancer occurrence and dairy product consumption in general, with no difference between fermented and unfermented products. This article argues that yogurt and other fermented milk products provide favorable health outcomes beyond the milk from which these products are made and that consumption of these products should be encouraged as part of national dietary guidelines. Systematic review registration: PROSPERO registration no. CRD42017068953.

Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract.

Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two ß-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway.IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

Probiotic Bifidobacterium strains and galactooligosaccharides improve intestinal barrier function in obese adults but show no synergism when used together as synbiotics.

BACKGROUND: One way to improve both the ecological performance and functionality of probiotic bacteria is by combining them with a prebiotic in the form of a synbiotic. However, the degree to which such synbiotic formulations improve probiotic strain functionality in humans has not been tested systematically. Our goal was to use a randomized, double-blind, placebo-controlled, parallel-arm clinical trial in obese humans to compare the ecological and physiological impact of the prebiotic galactooligosaccharides (GOS) and the probiotic strains Bifidobacterium adolescentis IVS-1 (autochthonous and selected via in vivo selection) and Bifidobacterium lactis BB-12 (commercial probiotic allochthonous to the human gut) when used on their own or as synbiotic combinations. After 3 weeks of consumption, strain-specific quantitative real-time PCR and 16S rRNA gene sequencing were performed on fecal samples to assess changes in the microbiota. Intestinal permeability was determined by measuring sugar recovery in urine by GC after consumption of a sugar mixture. Serum-based endotoxin exposure was also assessed. RESULTS: IVS-1 reached significantly higher cell numbers in fecal samples than BB-12 (P < 0.01) and, remarkably, its administration induced an increase in total bifidobacteria that was comparable to that of GOS. Although GOS showed a clear bifidogenic effect on the resident gut microbiota, both probiotic strains showed only a non-significant trend of higher fecal cell numbers when administered with GOS. Post-aspirin sucralose:lactulose ratios were reduced in groups IVS-1 (P = 0.050), IVS-1 + GOS (P = 0.022), and GOS (P = 0.010), while sucralose excretion was reduced with BB-12 (P = 0.002) and GOS (P = 0.020), indicating improvements in colonic permeability but no synergistic effects. No changes in markers of endotoxemia were observed. CONCLUSION: This study demonstrated that "autochthony" of the probiotic strain has a larger effect on ecological performance than the provision of a prebiotic substrate, likely due to competitive interactions with members of the resident microbiota. Although the synbiotic combinations tested in this study did not demonstrate functional synergism, our findings clearly showed that the pro- and prebiotic components by themselves improved markers of colonic permeability, providing a rational for their use in pathologies with an underlying leakiness of the gut.

Synbiotics for Improved Human Health: Recent Developments, Challenges, and Opportunities.

Research on combining pro- and prebiotics as synbiotics to enhance human and animal health has accelerated in the past 10 years, including many clinical trials that have assessed a diverse range of synbiotic formulations. In this review, we summarize these studies as well as the commercial applications of synbiotics that are available. In particular, we critically assess the claimed health benefits of synbiotic applications and the ecological and therapeutic factors to consider when designing synbiotics and discuss the implications of these concepts for future research in this field.

Galactooligosaccharide supplementation provides protection against Citrobacter rodentium-induced colitis without limiting pathogen burden.

Many enteric pathogens, including Salmonella and enteropathogenic and enterohemorrhagic Escherichia coli, express adhesins that recognize and bind to carbohydrate moieties expressed on epithelial cells. An attractive strategy for inhibiting bacterial adherence employs molecules that mimic these epithelial binding sites. Prebiotic oligosaccharides are non-digestible, fermentable fibres capable of modulating the gut microbiota. Moreover, they may act as molecular decoys that competitively inhibit adherence of pathogens to host cells. In particular, galactooligosaccharides (GOS) and other prebiotic fibres have been shown to inhibit pathogen adherence to epithelial cells in vitro. In the present study, we determined the ability of prophylactic GOS administration to reduce enteric pathogen adherence both in vitro and in vivo as well as protect against intestinal inflammation. GOS supplementation significantly reduced the adherence of the epithelial-adherent murine bacterial pathogen Citrobacter rodentium in a dose-dependent manner to the surface of epithelial cells in vitro. A 1- to 2-log reduction in bacterial adherence was observed at the lowest and highest doses tested, respectively. However, mouse studies revealed that treatment with GOS neither reduced the adherence of C. rodentium to the distal colon nor decreased its dissemination to systemic organs. Despite the absence of adherence inhibition, colonic disease scores for GOS-treated, C. rodentium-infected mice were significantly lower than those of untreated C. rodentium-infected animals (P=0.028). Together, these data suggest that GOS has a direct protective effect in ameliorating disease severity following C. rodentium infection through an anti-adherence-independent mechanism.

Subgingival Microbiome Colonization and Cytokine Production during Early Dental Implant Healing.

Little is known about longitudinal development of the peri-implant subgingival microbiome and cytokine production as a new sulcus forms after dental implant placement. Therefore, the purpose of this observational study was to evaluate simultaneous longitudinal changes in the oral microbiome and cytokine production in the developing peri-implant sulcus compared to control natural teeth. Four and 12 weeks after implant placement and abutment connection, a dental implant and a natural tooth were sampled in 25 patients for subgingival plaque and gingival crevicular fluid (GCF [around teeth] and peri-implant crevicular fluid [PICF] around implants). DNA from plaque samples was extracted and sequenced using Illumina-based 16S rRNA sequencing. GCF and PICF samples were analyzed using a customized Milliplex human cytokine and chemokine magnetic bead panel. Beta diversity analysis revealed that natural teeth and implants had similar subgingival microbiomes, while teeth had greater alpha diversity than implants. At the genus level, however, few differences were noted between teeth and dental implants over 12 weeks. Specifically, Actinomyces and Selenomonas were significantly elevated around teeth versus dental implants at both 4 weeks and 12 weeks, while Corynebacterium and Campylobacter were significantly elevated only at 4 weeks around teeth. The only difference between PICF and GCF biomarkers was significantly elevated granulocyte-macrophage colony-stimulating factor levels around teeth versus dental implants at the 4-week visit. The subgingival microbiome and cytokine production were similar between teeth and implants during early healing, suggesting that these profiles are driven by the patient following dental implant placement and are not determined by anatomical niche. IMPORTANCE Dental implants are a common treatment option offered to patients for tooth replacement. However, little is known regarding initial colonization of the subgingival microbiome and simultaneous longitudinal cytokine production in humans during the early healing phase following implant placement. We report findings from an in vivo study that assessed initial colonization of the subgingival microbiome and concomitant early cytokine production in a newly formed anatomical space, namely, an implant sulcus. This approach may be useful in future interventional studies to influence dental implant success. Our data showed that the subgingival microbiome and cytokine profile were similar for control natural teeth and dental implants at both 4 and 12 weeks after implant placement. These data suggest that these profiles are driven by the patient and not by anatomical location (i.e., tooth versus dental implant).

Stable Engraftment of Bifidobacterium longum AH1206 in the Human Gut Depends on Individualized Features of the Resident Microbiome.

Live bacteria (such as probiotics) have long been used to modulate gut microbiota and human physiology, but their colonization is mostly transient. Conceptual understanding of the ecological principles as they apply to exogenously introduced microbes in gut ecosystems is lacking. We find that, when orally administered to humans, Bifidobacterium longum AH1206 stably persists in the gut of 30% of individuals for at least 6 months without causing gastrointestinal symptoms or impacting the composition of the resident gut microbiota. AH1206 engraftment was associated with low abundance of resident B. longum and underrepresentation of specific carbohydrate utilization genes in the pre-treatment microbiome. Thus, phylogenetic limiting and resource availability are two factors that control the niche opportunity for AH1206 colonization. These findings suggest that bacterial species and functional genes absent in the gut microbiome of individual humans can be reestablished, providing opportunities for precise and personalized microbiome reconstitution.

Prebiotics and synbiotics: dietary strategies for improving gut health.

PURPOSE OF REVIEW: A wide range of dietary carbohydrates, including prebiotic food ingredients, fermentable fibers, and milk oligosaccharides, are able to produce significant changes in the intestinal microbiota. These shifts in the microbial community are often characterized by increased levels of bifidobacteria and lactobacilli. More recent studies have revealed that species of Faecalibacterium, Akkermansia, and other less well studied members may also be enriched. We review the implications of these recent studies on future design of prebiotics and synbiotics to promote gastrointestinal health. RECENT FINDINGS: Investigations assessing the clinical outcomes associated with dietary modification of the gut microbiota have shown systemic as well as specific health benefits. Both prebiotic oligosaccharides comprised of a linear arrangement of simple sugars, as well as fiber-rich foods containing complex carbohydrates, have been used in these trials. However, individual variability and nonresponding study participants can make the outcome of dietary interventions less predictable. In contrast, synergistic synbiotics containing prebiotics that specifically stimulate a cognate probiotic provide additional options for personalized gut therapies. SUMMARY: This review describes recent research on how prebiotics and fermentable fibers can influence the gut microbiota and result in improvements to human health.

Prebiotics: why definitions matter.

The prebiotic concept was introduced twenty years ago, and despite several revisions to the original definition, the scientific community has continued to debate what it means to be a prebiotic. How prebiotics are defined is important not only for the scientific community, but also for regulatory agencies, the food industry, consumers and healthcare professionals. Recent developments in community-wide sequencing and glycomics have revealed that more complex interactions occur between putative prebiotic substrates and the gut microbiota than previously considered. A consensus among scientists on the most appropriate definition of a prebiotic is necessary to enable continued use of the term.

Characterization of the ecological role of genes mediating acid resistance in Lactobacillus reuteri during colonization of the gastrointestinal tract.

Rodent-derived strains of Lactobacillus reuteri densely colonize the forestomach of mice and possess several genes whose predicted functions constitute adaptations towards an acidic environment. The objective of this study was to systematically determine which genes of L. reuteri 100-23 contribute to tolerance towards host gastric acid secretion. Genes predicted to be involved in acid resistance were inactivated, and their contribution to survival under acidic conditions was confirmed in model gastric juice. Fitness of five mutants that showed impaired in vitro acid resistance were then compared through competition experiments in ex-germ-free mice that were either treated with omeprazole, a proton-pump inhibitor that suppresses acid secretion in the stomach, or left untreated. This analysis revealed that the urease cluster was the predominant factor in mediating resistance to gastric acid production. Population levels of the mutant, which were substantially decreased in untreated mice, were almost completely restored through omeprazole, demonstrating that urease production in L. reuteri is mainly devoted to overcome gastric acid. The findings provide novel information on the mechanisms by which L. reuteri colonizes its gastric niche and demonstrate that in silico gene predictions and in vitro tests have limitations for predicting the ecological functions of colonization factors in bacterial symbionts.

Adherence Reduction of Campylobacter jejuni and Campylobacter coli Strains to HEp-2 Cells by Mannan Oligosaccharides and a High-Molecular-Weight Component of Cranberry Extract.

Campylobacter infections are a leading cause of human bacterial gastroenteritis in the United States and are a major cause of diarrheal disease throughout the world. Colonization and subsequent infection and invasion of Campylobacter require that the bacteria adhere to the surface of host cells. Agents that inhibit adherence could be used prophylactically to reduce Campylobacter carriage and infection. Mannan oligosaccharides (MOS) have been used as a feed supplement in livestock animals to improve performance and to replace growth-promoting antibiotics. However, MOS and other nondigestible oligosaccharides may also prevent pathogen colonization by inhibiting adherence in the gastrointestinal tract. In addition, plant extracts, including those derived from cranberries, have been shown to have antiadherence activity against pathogens. The goal of this study was to assess the ability of MOS and cranberry fractions to serve as antiadherence agents against strains of Campylobacter jejuni and Campylobacter coli. Adherence experiments were performed using HEp-2 cells. Significant reductions in adherence of C. jejuni 29438, C. jejuni 700819, C. jejuni 3329, and C. coli 43485 were observed in the presence of MOS (up to 40 mg/ml) and with a high-molecular-weight fraction of cranberry extract (up to 3 mg/ml). However, none of the tested materials reduced adherence of C. coli BAA-1061. No additive effect in adherence inhibition was observed for an MOS-cranberry blend. These results suggest that both components, MOS and cranberry, could be used to reduce Campylobacter colonization and carriage in livestock animals and potentially limit human exposure to this pathogen.

In vivo selection to identify bacterial strains with enhanced ecological performance in synbiotic applications.

One strategy for enhancing the establishment of probiotic bacteria in the human intestinal tract is via the parallel administration of a prebiotic, which is referred to as a synbiotic. Here we present a novel method that allows a rational selection of putative probiotic strains to be used in synbiotic applications: in vivo selection (IVS). This method consists of isolating candidate probiotic strains from fecal samples following enrichment with the respective prebiotic. To test the potential of IVS, we isolated bifidobacteria from human subjects who consumed increasing doses of galactooligosaccharides (GOS) for 9 weeks. A retrospective analysis of the fecal microbiota of one subject revealed an 8-fold enrichment in Bifidobacterium adolescentis strain IVS-1 during GOS administration. The functionality of GOS to support the establishment of IVS-1 in the gastrointestinal tract was then evaluated in rats administered the bacterial strain alone, the prebiotic alone, or the synbiotic combination. Strain-specific quantitative real-time PCR showed that the addition of GOS increased B. adolescentis IVS-1 abundance in the distal intestine by nearly 2 logs compared to rats receiving only the probiotic. Illumina 16S rRNA sequencing not only confirmed the increased establishment of IVS-1 in the intestine but also revealed that the strain was able to outcompete the resident Bifidobacterium population when provided with GOS. In conclusion, this study demonstrated that IVS can be used to successfully formulate a synergistic synbiotic that can substantially enhance the establishment and competitiveness of a putative probiotic strain in the gastrointestinal tract.

Production and in vitro fermentation of soluble, non-digestible, feruloylated oligo- and polysaccharides from maize and wheat brans.

High-pressure hydrothermal treatment of cereal bran results in fragmentation of the cell wall, releasing soluble, non-digestible, feruloylated oligo- and polysaccharides (FOPS), which may be beneficial to gut health. The objectives of this study were to (1) determine treatment temperatures for production of FOPS from maize bran and wheat bran and (2) determine the fermentation properties of partially purified FOPS from maize bran and wheat bran. FOPS were produced by heating bran and water (10%, w/v) in a high-pressure stirred reactor until the slurry reached 160-200 °C (in 10 °C increments). Final temperatures of 190 °C for maize bran and 200 °C for wheat bran resulted in the highest release of FOPS (49 and 50% of starting non-starch polysaccharide, respectively). Partial purification with ion exchange and dialysis resulted in a final product containing 63 and 57% total carbohydrate and 49 and 30% FOPS, respectively (other carbohydrate was starch). Following in vitro digestion (to remove starch), in vitro fermentation revealed that wheat FOPS were more bifidogenic than maize FOPS. However, maize FOPS led to continual production of short-chain fatty acid (SCFA), resulting in the highest SCFA and butyrate production at the end of the fermentation. In addition, maize FOPS showed significantly higher antioxidant activity than wheat FOPS. This study identified a process to produce FOPS from maize bran and wheat bran and showed that, considering the overall beneficial effects, FOPS from maize bran may exhibit enhanced benefits on gut health compared to those of wheat bran.

Adherence inhibition of enteropathogenic Escherichia coli by chitooligosaccharides with specific degrees of acetylation and polymerization.

Some oligosaccharides are known to act as molecular decoys by inhibiting pathogen adherence to epithelial cells. The present study was aimed at analyzing whether chitooligosaccharides (CHOS), that is, oligomers of D-glucosamine and N-acetyl-D-glucosamine, have such antiadherence activity. CHOS of varied degree of polymerization (DP) and fraction of acetylation (F(A)) were produced. Adherence of enteropathogenic Escherichia coli (EPEC) to the surface of a human HEp-2 cell line was determined in the absence or presence of the various CHOS fractions. Adherence was assessed by microscopic counting and image analysis of bacterial clusters and cells. The results showed that all CHOS fractions inhibited adherence of EPEC to HEp-2 cells. Hydrolysates with lower F(A) were more effective at reducing adherence. This effect is greater than that obtained with other oligosaccharides, such as galactooligosaccharides, applied at the same concentrations.

Xylo-oligosaccharides inhibit pathogen adhesion to enterocytes in vitro.

We previously reported that the non-digestible carbohydrates inulin and apple pectin promoted Listeria monocytogenes infection in guinea pigs, whereas xylo- and galacto-oligosaccharides (XOS and GOS), prevented infection by this pathogen. In the present study, mechanisms that could explain the previous in vivo observations were explored. Mixing bacterial cultures with XOS significantly (P < 0.05) decreased the ability of two out of three strains of L. monocytogenes to adhere to Caco-2 cells. Additionally, 2 h incubation with XOS followed by washing of the bacteria significantly (P < 0.05) decreased the ability of all three strains to adhere to Caco-2 cells. Consistently, expression of the adhesion-relevant genes inlA and lap was reduced by the presence of XOS. The observation that XOS inhibit the adhesion of Listeria to the intestinal epithelium in vitro may explain the reported preventive effect of XOS on Listeria infection in guinea pigs in vivo, while the preventive effect of GOS was not explicable by the assays chosen here.

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.

Adherence, anti-adherence, and oligosaccharides preventing pathogens from sticking to the host.

For many pathogenic bacteria, infections are initiated only after the organism has first adhered to the host cell surface. If adherence can be inhibited, then the subsequent infection can also be inhibited. This approach forms the basis of anti-adherence strategies, which have been devised to prevent a variety of bacterial infections. In this chapter, the molecular basis by which respiratory, urinary, and gastrointestinal tract pathogens adhere to host cells will be described. The five general types of anti-adherence agents will also be reviewed. The most well-studied are the receptor analogs, which include oligosaccharides produced synthetically or derived from natural sources, including milk, berries, and other plants. Their ability to inhibit pathogen adherence may lead to development of novel, food-grade anti-infective agents that are inexpensive and safe.

Functional analysis of the fructooligosaccharide utilization operon in Lactobacillus paracasei 1195.

The fosABCDXE operon encodes components of a putative fructose/mannose phosphoenolpyruvate-dependent phosphotransferase system and a beta-fructosidase precursor (FosE) that are involved in the fructooligosaccharide (FOS) utilization pathway of Lactobacillus paracasei 1195. The presence of an N-terminal signal peptide sequence and an LPQAG cell wall anchor motif in the C-terminal region of the deduced FosE precursor amino acid sequence predicted that the enzyme is cell wall associated, indicating that FOS may be hydrolyzed extracellularly. In this study, cell fractionation experiments demonstrated that the FOS hydrolysis activity was present exclusively in the cell wall extract of L. paracasei previously grown on FOS. In contrast, no measurable FOS hydrolysis activity was detected in the cell wall extract from the isogenic fosE mutant. Induction of beta-fructosidase activity was observed when cells were grown on FOS, inulin, sucrose, or fructose but not when cells were grown on glucose. A diauxic growth pattern was observed when cells were grown on FOS in the presence of limiting glucose (0.1%). Analysis of the culture supernatant revealed that glucose was consumed first, followed by the longer-chain FOS species. Transcription analysis further showed that the fos operon was expressed only after glucose was depleted in the medium. Expression of fosE in a non-FOS-fermenting strain, Lactobacillus rhamnosus GG, enabled the recombinant strain to metabolize FOS, inulin, sucrose, and levan.

Identification of a putative operon involved in fructooligosaccharide utilization by Lactobacillus paracasei.

The growth and activity of some Lactobacillus and Bifidobacterium strains are stimulated by the presence of nondigestible fructooligosaccharides (FOS), which are selectively fermented by specific intestinal bacteria. Consumption of FOS, therefore, enriches for those bacteria that possess metabolic pathways necessary for FOS metabolism. In this study, a DNA microarray consisting of 7,680 random genomic library fragments of Lactobacillus paracasei 1195 was used to examine genes involved in the utilization of FOS in this organism. Differential expression profiles between cells grown on FOS and those grown on glucose provided a basis for identifying genes specifically induced by FOS. Several of the FOS-induced genes shared sequence identity with genes encoding beta-fructosidases and components of phosphoenolpyruvate-dependent phosphotransferase systems (PTS). These genes were organized in a putative operon, designated the fos operon, that may play an essential role in FOS utilization. The complete 7,631-bp nucleotide sequence of the putative fos operon was determined and consists of fosABCDXE genes, which encode a putative fructose/mannose PTS (FosABCDX) and a beta-fructosidase precursor (FosE). The latter contains an N-terminal signal peptide sequence and cell wall sorting signals at the C-terminal region, suggesting its localization at the cell wall. Inactivation of the fosE gene led to impaired growth on FOS and other beta-fructose-linked carbohydrates. Transcriptional analysis by reverse transcriptase PCR suggested that fosABCDXE was cotranscribed as a single mRNA during growth on FOS. Expression array analysis revealed that when glucose was added to FOS-grown cells, transcription of the FOS-induced genes was repressed, indicating that FOS metabolism is subject to catabolite regulation.

Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells.

Prebiotic oligosaccharides are thought to provide beneficial effects in the gastrointestinal tract of humans and animals by stimulating growth of selected members of the intestinal microflora. Another means by which prebiotic oligosaccharides may confer health benefits is via their antiadhesive activity. Specifically, these oligosaccharides may directly inhibit infections by enteric pathogens due to their ability to act as structural mimics of the pathogen binding sites that coat the surface of gastrointestinal epithelial cells. In this study, the ability of commercial prebiotics to inhibit attachment of microcolony-forming enteropathogenic Escherichia coli (EPEC) was investigated. The adherence of EPEC strain E2348/69 on HEp-2 and Caco-2 cells, in the presence of fructooligosaccharides, inulin, galactooligosaccharides (GOS), lactulose, and raffinose was determined by cultural enumeration and microscopy. Purified GOS exhibited the greatest adherence inhibition on both HEp-2 and Caco-2 cells, reducing the adherence of EPEC by 65 and 70%, respectively. In addition, the average number of bacteria per microcolony was significantly reduced from 14 to 4 when GOS was present. Adherence inhibition by GOS was dose dependent, reaching a maximum at 16 mg/ml. When GOS was added to adhered EPEC cells, no displacement was observed. The expression of BfpA, a bundle-forming-pilus protein involved in localized adherence, was not affected by GOS, indicating that adherence inhibition was not due to the absence of this adherence factor. In addition, GOS did not affect autoaggregation. These observations suggest that some prebiotic oligosaccharides may have antiadhesive activity and directly inhibit the adherence of pathogens to the host epithelial cell surface.