Michael J. Miller

The health benefits of dietary fiber: Beyond the usual suspects of type 2 diabetes mellitus, cardiovascular disease and colon cancer

Dietary fiber (DF) is deemed to be a key component in healthy eating. DF is not a static collection of undigestible plant materials that pass untouched or unencumbered through the gastrointestinal (GI) tract; instead, DFs are a vast array of complex saccharide-based molecules that can bind potential nutrients and nutrient precursors to prevent their absorption. Some DFs are fermentable, and the GI tract catabolism leads to the generation of various bioactive materials, such as short-chain fatty acids (SCFAs), that can markedly augment the GI tract biomass and change the composition of the GI tract flora. The health benefits of DFs include the prevention and mitigation of type 2 diabetes mellitus, cardiovascular disease and colon cancer. By modulating food ingestion, digestion, absorption and metabolism, DFs reduce the risk of hyperlipidemia, hypercholesterolemia and hyperglycemia. Emerging research has begun to investigate the role of DFs in immunomodulation. If substantiated, DFs could facilitate many biologic processes, including infection prevention and the improvement of mood and memory. This review describes the accepted physiologic functions of DFs and explores their new potential immune-based actions.

Lactobacillus adhesion to mucus.

Mucus provides protective functions in the gastrointestinal tract and plays an important role in the adhesion of microorganisms to host surfaces. Mucin glycoproteins polymerize, forming a framework to which certain microbial populations can adhere, including probiotic Lactobacillus species. Numerous mechanisms for adhesion to mucus have been discovered in lactobacilli, including partially characterized mucus binding proteins. These mechanisms vary in importance with the in vitro models studied, which could significantly affect the perceived probiotic potential of the organisms. Understanding the nature of mucus-microbe interactions could be the key to elucidating the mechanisms of probiotic adhesion within the host.

Analysis of the Genome Sequence of Lactobacillus gasseri ATCC 33323 Reveals the Molecular Basis of an Autochthonous Intestinal Organism

ABSTRACT This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.

Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants.

BackgroundIndividuals with inactive alleles of the fucosyltransferase 2 gene (FUT2; termed the ‘secretor’ gene) are common in many populations. Some members of the genus Bifidobacterium, common infant gut commensals, are known to consume 2′-fucosylated glycans found in the breast milk of secretor mothers. We investigated the effects of maternal secretor status on the developing infant microbiota with a special emphasis on bifidobacterial species abundance.ResultsOn average, bifidobacteria were established earlier and more often in infants fed by secretor mothers than in infants fed by non-secretor mothers. In secretor-fed infants, the relative abundance of the Bifidobacterium longum group was most strongly correlated with high percentages of the order Bifidobacteriales. Conversely, in non-secretor-fed infants, Bifidobacterium breve was positively correlated with Bifidobacteriales, while the B. longum group was negatively correlated. A higher percentage of bifidobacteria isolated from secretor-fed infants consumed 2′-fucosyllactose. Infant feces with high levels of bifidobacteria had lower milk oligosaccharide levels in the feces and higher amounts of lactate. Furthermore, feces containing different bifidobacterial species possessed differing amounts of oligosaccharides, suggesting differential consumption in situ.ConclusionsInfants fed by non-secretor mothers are delayed in the establishment of a bifidobacteria-laden microbiota. This delay may be due to difficulties in the infant acquiring a species of bifidobacteria able to consume the specific milk oligosaccharides delivered by the mother. This work provides mechanistic insight into how milk glycans enrich specific beneficial bacterial populations in infants and reveals clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants.

Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in Aspergillus parasiticus

ABSTRACT The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-β-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.

Sensory and microbiological quality of yogurt drinks with prebiotics and probiotics

The popularity of dairy products fortified with prebiotics and probiotics continues to increase as consumers desire flavorful foods that will fulfill their health needs. Our objectives were to assess the sensory profile of drinkable yogurts made with prebiotics and probiotics and to determine the viability of the probiotics in the yogurt drink over the duration of storage. Thirteen trained descriptive panelists evaluated 10 yogurt drinks on a 16-point category scale. Three selected prebiotics, soluble corn fiber, polydextrose, and chicory inulin, were each present individually at an amount to claim an excellent source of fiber (5 g of fiber/serving) or a good source of fiber (2.5 g of fiber/serving) in 6 different yogurt drinks. Three additional yogurt drinks contained 5 g of each of the separate prebiotics along with a mixture of the selected probiotics (Bifidobacterium lactis Bb-12 and Lactobacillus acidophilus LA-5). A control sample with no prebiotics or probiotics was also included in the experimental design. Data were analyzed by ANOVA, Fishers least significant difference, and principal component analysis. Survival of the probiotics in the yogurt drinks during a 30-d refrigerated storage period was also analyzed. Results showed that clover honey aroma, buttermilk aroma, butter aroma, sweetness, sourness, chalky mouthfeel, and viscosity were identified as significant attributes in the yogurt drinks. Total variance explained by the principal component analysis biplot of factors 1 and 2 was 65%, which showed yogurt drinks with soluble corn fiber and inulin varying by the sweet versus sour attributes and yogurt drinks with polydextrose varying by the mouthfeel attributes. The viability study determined a 2- to 3-log decrease in the survival of probiotics in all of the yogurt treatments during a 30-d refrigerated storage period. Based on the results of the current study, only the polydextrose treatment would be an acceptable vehicle to deliver the probiotic health effects at the end of the 30-d storage period.

Methylphenidate prevents high-fat diet (HFD)-induced learning/memory impairment in juvenile mice

The prevalence of childhood obesity has risen dramatically and coincident with this upsurge is a growth in adverse childhood psychological conditions including impulsivity, depression, anxiety and attention deficit/hyperactive disorder (ADHD). Due to confounds that exist when determining causality of childhood behavioral perturbations, controversy remains as to whether overnutrition and/or childhood obesity is important. Therefore, we examined juvenile mice to determine if biobehaviors were impacted by a short-term feeding (1-3wks) of a high-fat diet (HFD). After 1wk of a HFD feeding, mouse burrowing and spontaneous wheel running were increased while mouse exploration of the open quadrants of a zero maze, perfect alternations in a Y-maze and recognition of a novel object were impaired. Examination of mouse cortex, hippocampus and hypothalamus for dopamine and its metabolites demonstrated increased homovanillic acid (HVA) concentrations in the hippocampus and cortex that were associated with decreased cortical BDNF gene expression. In contrast, pro-inflammatory cytokine gene transcripts and serum IL-1α, IL-1β, TNF-α and IL-6 were unaffected by the short-term HFD feeding. Administration to mice of the psychostimulant methylphenidate prevented HFD-dependent impairment of learning/memory. HFD learning/memory impairment was not inhibited by the anti-depressants desipramine or reboxetine nor was it blocked in IDO or IL-1R1 knockout mice. In sum, a HFD rapidly impacts dopamine metabolism in the brain appearing to trigger anxiety-like behaviors and learning/memory impairments prior to the onset of weight gain and/or pre-diabetes. Thus, overnutrition due to fats may be central to childhood psychological perturbations such as anxiety and ADHD.

Human milk oligosaccharides shorten rotavirus-induced diarrhea and modulate piglet mucosal immunity and colonic microbiota.

The impact of human milk oligosaccharides (HMO) on mucosal immunity, gut microbiota and response to rotavirus (RV) infection was investigated in the piglet model. Newborn piglets were fed with formula alone (FF) or formula supplemented with 4 g l−1 HMO (HMO) or a prebiotic mixture of 9:1 short-chain galactooligosaccharides (3.6 g l−1) and long-chain fructooligosaccharides (0.4 g l−1) (PRE) (n=19–21 per group) for 15 days. Piglets (n=7–8) in each dietary group were orally infected with porcine rotavirus (RV) OSU strain on d10, and stool consistency was assessed daily. Blood, small intestine and colonic contents were collected at day 15. Serum RV-specific antibody concentrations, intestinal histomorphology, RV non-structural protein-4 (NSP4) and cytokine mRNA expression were assessed. Colonic content pH, dry matter (DM) and short-chain fatty acid concentrations were measured. Ascending colonic microbiota was analyzed by 16S rRNA gene v1-3 region pyrosequencing. HMO- and PRE-fed groups had shorter duration of diarrhea than FF piglets. Infection changed intestinal histomorphology, increased serum RV-specific antibody response and intestinal RV NSP4 expression, and modulated ileal cytokine expression. HMO enhanced T helper type 1 (interferon-gamma) and anti-inflammatory (interleukin-10) cytokines in the ileum, while prebiotics promoted RV-specific immunoglobulin M response to the infection. RV infection and HMO supplementation altered intraluminal environment and gut microbiota. HMO increased pH and lowered DM of colonic contents and enhanced the abundance of unclassified Lachnospiraceae, which contains numerous butyrate-producing bacteria. In conclusion, HMO and prebiotics did not prevent the onset of RV infection but reduced the duration of RV-induced diarrhea in piglets, in part, by modulating colonic microbiota and immune response to RV infection.

Construction of vectors for inducible and constitutive gene expression in Lactobacillus

Microarray analysis of the genome of Lactobacillus acidophilus identified a number of operons that were differentially expressed in response to carbohydrate source or constitutively expressed regardless of carbohydrate source. These included operons implicated in the transport and catabolism of fructooligosaccharides (FOS), lactose (lac), trehalose (tre) and genes directing glycolysis. Analysis of these operons identified a number of putative promoter and repressor elements, which were used to construct a series of expression vectors for use in lactobacilli, based on the broad host range pWV01 replicon. A β‐glucuronidase (GusA3) reporter gene was cloned into each vector to characterize expression from each promoter. GUS reporter assays showed FOS, lac and tre based vectors to be highly inducible by their specific carbohydrate and repressed by glucose. Additionally, a construct based on the phosphoglycerate mutase (pgm) promoter was constitutively highly expressed. To demonstrate the potential utility of these vectors, we constructed a plasmid for the overexpression of the oxalate degradation pathway (Frc and Oxc) of L. acidophilus NCFM. This construct was able to improve oxalate degradation by L. gasseri ATCC 33323 and compliment a L. acidophilus oxalate‐deficient mutant. Development of these expression vectors could support several novel applications, including the expression of enzymes, proteins, vaccines and biotherapeutics by intestinal lactobacilli.

Drivers of liking for yogurt drinks with prebiotics and probiotics

Several studies have addressed the sensory properties of yogurt. However, as the market for yogurt continues to expand and new varieties of yogurt with novel ingredients emerge, additional sensory tests are needed to ensure the quality of the products. Three selected prebiotics, soluble corn fiber, polydextrose, and chicory inulin, were each added at an excellent source of fiber (5 g fiber/serving) or a good fiber source (2.5 g fiber/serving) levels into a yogurt drink base. Three additional yogurt drinks contained 5 g of each of the separate prebiotics along with a mixture of probiotics (Bifidobacterium lactis Bb-12 and Lactobacillus acidophilus LA-5). A control sample with no prebiotics or probiotics was also included in the experimental design. Yogurt drinks were evaluated by 110 consumers for overall acceptance, acceptance of aroma, appearance, taste, and texture, and purchase intent. Demographic information pertaining to consumer knowledge of prebiotics and probiotics was collected. Consumer data were correlated with previously obtained descriptive analysis data to identify drivers of liking. Data were analyzed by analysis of variance (ANOVA), Fishers least significant difference (LSD), cluster analysis, internal preference mapping, and external preference mapping. Total variance explained by the internal and external preference maps were 32.2% and 64.6%, respectively, which showed higher levels of the prebiotics with probiotics drove consumer liking compared to lower levels without probiotics. In terms of ingredients added, chicory inulin and polydextrose were preferred over soluble corn fiber. Yogurt drinks with these prebiotics included and probiotics were characterized by a medium level of sweetness and high viscosity. Development of new prebiotic and probiotic containing drinkable yogurts should strive for a medium level of sweetness and high viscosity for maximum consumer acceptance.