Dietary Modulation of the Human Gut Microbiota and Metabolome with Flaxseed Preparations.

Flaxseeds are typically consumed either as whole flaxseed, ground flaxseed, flaxseed oil, partially defatted flaxseed meal, or as a milk alternative. They are considered a rich source of vitamins, minerals, proteins and peptides, lipids, carbohydrates, lignans, and dietary fiber, which have shown hypolipidemic, antiatherogenic, anticholesterolemic, and anti-inflammatory property activity. Here, an in vitro batch culture model was used to investigate the influence of whole milled flaxseed and partially defatted milled flaxseed press cake on the gut microbiota and the liberation of flaxseed bioactives. Microbial communities were profiled using 16S rRNA gene-based high-throughput sequencing with targeted mass spectrometry measuring lignan, cyclolinopeptide, and bile acid content and HPLC for short-chain fatty acid profiles. Flaxseed supplementation decreased gut microbiota richness with Firmicutes, Proteobacteria, and Bacteroidetes becoming the predominant phyla. Secoisolariciresinol, enterodiol, and enterolactone were rapidly produced with acetic acid, butyric acid, and propionic acid being the predominant acids after 24 h of fermentation. The flaxseed press cake and whole flaxseed were equivalent in microbiota changes and functionality. However, press cake may be superior as a functional additive in a variety of foods in terms of consumer acceptance as it would be more resistant to oxidative changes.

Virulence Factors of Enteric Pathogenic Escherichia coli: A Review.

Escherichia coli are remarkably versatile microorganisms and important members of the normal intestinal microbiota of humans and animals. This harmless commensal organism can acquire a mixture of comprehensive mobile genetic elements that contain genes encoding virulence factors, becoming an emerging human pathogen capable of causing a broad spectrum of intestinal and extraintestinal diseases. Nine definite enteric E. coli pathotypes have been well characterized, causing diseases ranging from various gastrointestinal disorders to urinary tract infections. These pathotypes employ many virulence factors and effectors subverting the functions of host cells to mediate their virulence and pathogenesis. This review summarizes new developments in our understanding of diverse virulence factors associated with encoding genes used by different pathotypes of enteric pathogenic E. coli to cause intestinal and extraintestinal diseases in humans.

Enzymatic Modification of Native Chitin and Conversion to Specialty Chemical Products.

: Chitin is one of the most abundant biomolecules on earth, occurring in crustacean shells and cell walls of fungi. While the polysaccharide is threatening to pollute coastal ecosystems in the form of accumulating shell-waste, it has the potential to be converted into highly profitable derivatives with applications in medicine, biotechnology, and wastewater treatment, among others. Traditionally this is still mostly done by the employment of aggressive chemicals, yielding low quality while producing toxic by-products. In the last decades, the enzymatic conversion of chitin has been on the rise, albeit still not on the same level of cost-effectiveness compared to the traditional methods due to its multi-step character. Another severe drawback of the biotechnological approach is the highly ordered structure of chitin, which renders it nigh impossible for most glycosidic hydrolases to act upon. So far, only the Auxiliary Activity 10 family (AA10), including lytic polysaccharide monooxygenases (LPMOs), is known to hydrolyse native recalcitrant chitin, which spares the expensive first step of chemical or mechanical pre-treatment to enlarge the substrate surface. The main advantages of enzymatic conversion of chitin over conventional chemical methods are the biocompability and, more strikingly, the higher product specificity, product quality, and yield of the process. Products with a higher Mw due to no unspecific depolymerisation besides an exactly defined degree and pattern of acetylation can be yielded. This provides a new toolset of thousands of new chitin and chitosan derivatives, as the physio-chemical properties can be modified according to the desired application. This review aims to provide an overview of the biotechnological tools currently at hand, as well as challenges and crucial steps to achieve the long-term goal of enzymatic conversion of native chitin into specialty chemical products.

Phylogenetic analysis and antibiotic resistance of Shigella sonnei isolates.

Shigellosis is one of the most important gastric infections caused by different species of Shigella, and has been regarded as a serious threat to public health. Lineage/sublineage profile of Shigella sonnei is strongly associated with the antibiotic resistance and population structure of this pathogen. In this study, we determined the phylogeny and antibiotic resistance profiles of S. sonnei strains, isolated from 1246 stool and 580 food samples, using multiplex PCR-HRMA genotyping and Kirby-Bauer disk diffusion methods, respectively. A total of 64 S. sonnei strains were isolated (13 food and 51 clinical isolates). Multiplex PCR-HMR assay was able to differentiate the lineages II and III, and sublineages IIIb and IIIc strains successfully considering the definite melting curves and temperatures. Lineage I and sublineage IIIa strain were not isolated in this study. We also demonstrated that most of the S. sonnei strains isolated from both food and clinical samples clustered within the lineage III and sublineage IIIc. Resistance against trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, and streptomycin antibiotics were the most prevalent phenotypes among the S. sonnei lineage III and sublineage IIIc strains.

Fermentative Liberation of Ellagic Acid from Walnut Press Cake Ellagitannins.

Oil is extracted from walnut leaves behind large quantities of defatted press cake that is still rich in valuable nutrients. Aspergillus oryzae and Rhizopus oligosporus, two molds traditionally used in Asia, have the necessary enzymes to use the nutrients in the walnut press cake. Walnuts and the press cake contain ellagitannins, known as precursors for ellagic acid and urolithins. In this study, experiments to optimize the solid-state fermentation of walnut press cake were performed in order to liberate ellagic acid from ellagitannins. Extracts of fermented products were then analyzed with an HPLC-DAD to measure the liberation of ellagic acid from ellagitannins. Good growth of R. oligosporus and A. oryzae mycelia on the walnut press cake was observed. A single mold culture was subjected to a hydration of 0.8 mL/g, an addition of 37.5 mmol/kg acetic acid (AA) and 1% NaCl, and an incubation temperature of 25 °C; these were observed to be good conditions for solid-state fermentation for walnut press cake. The highest ellagic acid concentration was obtained at 48 h. At 72 h, degradation dominated the liberation of ellagic acid.

A new prokaryotic farnesyldiphosphate synthase from the octocoral Eunicea fusca: differential display, inverse PCR, cloning, and characterization.

We recently reported that the biosynthesis of fuscol, a diterpene from the octocoral Eunicea fusca, is inducible by the application of plant signaling factors such as salicylic acid to the coral's algal symbiont. In this study, an mRNA differential display approach has been employed with the dinoflagellate symbiont of this octocoral which has led to the isolation of a farnesyldiphosphate synthase (FPPS) that was transcriptionally activated under conditions that led to an induction of fuscol biosynthesis. Using a degenerate primer based on the aspartate-rich motifs found in prenylsynthases and a cassette ligation strategy, we report the cloning of the complete FPPS associated with the E. fusca dinoflagellate symbiont Symbiodinium sp. The protein exhibited the enzymatic properties associated with FPPS, namely, the synthesis of farnesyl diphosphate from geranyldiphosphate and isopentenyl diphosphate. The amino acid sequence of this FPPS has a high sequence similarity (82%) to known archaeal isoprenyl diphosphate synthases. This is the first description of a prokaryotic FPPS derived from a marine source.

Diversity of the bacterial communities associated with the azooxanthellate deep water octocorals Leptogorgia minimata, Iciligorgia schrammi, and Swiftia exertia.

This study examined the microbiota associated with the marine azooxanthellate octocorals Leptogorgia minimata, Swiftia exertia, and Iciligorgia schrammi collected from moderate depths (45 m). Traditional aerobic plate culture, fluorescence in situ hybridization (FISH), and molecular identification of the 16S rDNA region were used for this purpose. In general, cultures were found to be selective for Gammaproteobacteria, Alphaproteobacteria, and Firmicutes. Interestingly, FISH counts for Firmicutes in the whole coral (holobiont) were near the detection limit of this assay, representing less than 6% of the total detectable microbiota in all counts. Proteobacteria, especially Alpha- and Gammaproteobacteria, made up the majority of the total microbiota in the holobionts. In addition, the absence of zooxanthellae in these three corals was confirmed by the use of polymerase chain reaction (PCR) and dinoflagellate-specific primers, and spectrophotometric chlorophyll pigment measurements. No evidence of zooxanthellae could be found in any of the corals by either of these techniques. This is the first study examining the microbiota marine octocorals, which grow at moderate depth (40 to 100 m) in the absence of direct sunlight.

Isolation of proteolytic bacteria from mealworm (Tenebrio molitor) exoskeletons to produce chitinous material.

The use of insects as a source of protein is becoming an important factor for feeding an increasing population. After protein extraction for food use, the insect exoskeleton may offer the possibility for the production of added value products. Here, the aim was to isolate bacteria from the surface of farmed mealworms (Tenebrio molitor Linnaeus, 1758) for the production of chitinous material from insect exoskeletons using microbial fermentation. Isolates were screened for proteases and acid production that may aid deproteination and demineralisation of insects through fermentation to produce chitin. Selected isolates were used single-step (isolated bacteria only) or two-step fermentations with Lactobacillus plantarum (DSM 20174). Two-step fermentations with isolates from mealworm exoskeletons resulted in a demineralisation of 97.9 and 98.5% from deproteinated mealworm fractions. Attenuated total reflectance-Fourier- transform infrared spectroscopy analysis showed that crude chitin was produced. However, further optimisation is needed before the process can be upscaled. This is, to our knowledge, the first report using microbial fermentation for the extraction of chitin from insects.

The effects of alpha-lactalbumin and glycomacropeptide on the association of CaCo-2 cells by enteropathogenic Escherichia coli, Salmonella typhimurium and Shigella flexneri.

Two milk components, alpha-lactalbumin (alpha-La) and glycomacropeptide (GMP) may inhibit intestinal infection/intoxification. (3)[H] thymidine-labeled enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium (ATCC 6994) or Shigella flexneri (ATCC 9199) were introduced to CaCo-2 cultures and their association with CaCo-2 cells was assessed. Undigested, pepsin-digested and pepsin- and pancreatin-digested alpha-lactalbumin and glycomacropeptide inhibited association. Thus, milk supplemented with alpha-lactalbumin and glycomacropeptide might be effective in inhibiting associations of the pathogens EPEC, Salmonella typhimurium, and Shigella flexneri to intestinal cells.

Effects of bovine alpha-lactalbumin and casein glycomacropeptide-enriched infant formulae on faecal microbiota in healthy term infants.

OBJECTIVE: Certain milk factors may promote the growth of a host-friendly gastrointestinal microbiota, for example, one that is predominated by bifidobacteria, a perceived health-promoting genus. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts who are believed to have a more diverse microbiota, which is similar to that of adults. The effects of formulas supplemented with 2 such ingredients from bovine milk, alpha-lactalbumin (alpha-lac) and casein glycomacropeptide (GMP), on gut flora were investigated in this study. PATIENTS AND METHODS: Six-week-old (4-8 wk), healthy term infants were randomised to a standard infant formula or 1 of 2 test formulae enriched in alpha-lac with higher or lower GMP until 6 months. Faecal bacteriology was determined by the culture-independent procedure fluorescence in situ hybridisation. RESULTS: There was a large fluctuation of bacterial counts within groups with no statistically significant differences between groups. Although all groups showed a predominance of bifidobacteria, breast-fed infants had a small temporary increase in counts. Other bacterial levels varied in formula-fed groups, which overall showed an adult-like faecal microflora. CONCLUSIONS: It can be speculated that a prebiotic effect for alpha-lac and GMP is achieved only with low starting populations of beneficial microbiota (eg, infants not initially breast-fed.

In vitro activity of commercial probiotic Lactobacillus strains against uropathogenic Escherichia coli.

Urinary tract infection (UTI) is one of the most prevalent infections in humans. In ≥80% of cases, the etiologic agents are strains of uropathogenic Escherichia coli (UPEC), which commonly reside in the gastrointestinal tract. Lactobacilli have been shown to prevent UTI reoccurrence by restoring the urogenital microbiota when administered vaginally or orally. The goal of this study was to determine if commercial probiotic Lactobacillus spp. reduce or clear UPEC in vitro. Results show that it is likely that lactobacilli may, in addition to restoring a healthy urogenital microbiota through acidification of their environment, also displace adhering UPEC and cause a reduction of infection.

Rate of establishing the gut microbiota in infancy has consequences for future health.

The gut of the human neonate is colonized rapidly after birth from an early sparse and highly distinct microbiota to a more adult-like and convergent state, within 1 to 3 years. The progression of colonizing bacterial species is non-random. During the first months of life several shifts commonly occur in the species prevalent in our guts. Although the sequential progression of these species is remarkably consistent across individuals and geographies, there is inter-individual variation in the rate of progression. Our study and others suggest that the rate is influenced by environmental factors, and influences our future health. In this article, we review our recent contribution to cataloging the developing infant gut microbiota alongside other important recent studies. We suggest testable hypotheses that arise from this synthesis.

Dynamics of infant gut microbiota are influenced by delivery mode and gestational duration and are associated with subsequent adiposity.

UNLABELLED: We found that the relatively simple microbiota of young infants shifts predictably to a more mature anaerobic microbiota during infancy and the dynamics of this shift are influenced by environmental factors. In this longitudinal study of 75 infants, we demonstrate high interindividual variability within the normal range of birth outcomes, especially in the rate of microbiota progression. Most had acquired a microbiota profile high in Bifidobacterium and Collinsella by 6 months of age, but the time point of this acquisition was later in infants delivered by caesarean section and those born after a shorter duration of gestation. Independently of the delivery mode and gestation duration, infants who acquired a profile high in Bifidobacterium and Collinsella at a later age had lower adiposity at 18 months of age. IMPORTANCE: This study shows that the acquisition of the early microbiota is strongly influenced by environmental factors such as the delivery mode and duration of gestation, even in healthy neonates. The composition of the early microbiota has been linked with long-lasting effects on health and disease. Here we show that the rate of acquisition of certain microbiota predicts adiposity at 18 months of age and so potentially the risk of later obesity.

The effect of proteolysis on the induction of cell death by monomeric alpha-lactalbumin.

α-Lactalbumin (α-la) is a major whey protein found in milk. Previous data suggested that α-la has antiproliferative effects in human adenocarcinoma cell lines such as Caco-2 and HT-29. However, the cell death inducing α-la was not a naturally occurring monomer but either a multimeric variant or an α-la:oleic acid complex (HAMLET/BAMLET). Proteolysis showed that both human and bovine α-la are susceptible to digestion. ELISA assays assessing cell death with the native undigested α-la fractions showed that undigested protein fractions did have a significant cell death effect on CaCo-2 cells. Bovine α-la was also more effective than human α-la. A reduction in activity corresponded with lower concentrations of the protein and partial digestion and fragmentation of the protein using trypsin and pepsin. This suggests that the tertiary structure is vital for the apoptotic effect.

The bacterial community of the lithistid sponge Discodermia spp. as determined by cultivation and culture-independent methods.

The marine lithistid sponge Discodermia spp. (Family Theonellidae) contains many types of associated bacteria visible in the mesohyl while biofilms cover the pinacoderm. This study determined the identity of bacteria associated with members of the genus Discodermia using microbial culture, 16S rRNA gene clone libraries and fluorescence in situ hybridization. Four samples of Discodermia spp. were collected at depths between 24-161 m near Grand Bahama Island and Cay Sal Bank, Bahamas. A total of 80 unique isolates and 94 different clone sequences from at least eight bacterial classes were obtained. It appeared that Discodermia spp. may have a core community of bacteria that is common to all sponges of this genus. Species of at least six different classes of bacteria were regularly found in most of the sponge specimens collected, irrespective of collection depth or location. This indicates that a diverse spectrum of bacteria is associated with lithistid sponges irrespective of the transient seawater community that enters the sponge.

Comparison of the anaerobic microbiota of deep-water Geodia spp. and sandy sediments in the Straits of Florida.

Marine sediments and sponges may show steep variations in redox potential, providing niches for both aerobic and anaerobic microorganisms. Geodia spp. and sediment specimens from the Straits of Florida were fixed using paraformaldehyde and 95% ethanol (v/v) for fluorescence in situ hybridization (FISH). In addition, homogenates of sponge and sediment samples were incubated anaerobically on various cysteine supplemented agars. FISH analysis showed a prominent similarity of microbiota in sediments and Geodia spp. samples. Furthermore, the presence of sulfate-reducing and annamox bacteria as well as other obligate anaerobic microorganisms in both Geodia spp. and sediment samples were also confirmed. Anaerobic cultures obtained from the homogenates allowed the isolation of a variety of facultative anaerobes, primarily Bacillus spp. and Vibrio spp. Obligate anaerobes such as Desulfovibrio spp. and Clostridium spp. were also found. We also provide the first evidence for a culturable marine member of the Chloroflexi, which may enter into symbiotic relationships with deep-water sponges such as Geodia spp. Resuspended sediment particles, may provide a source of microorganisms able to associate or form a symbiotic relationship with sponges.

Identification of the bacterial symbiont Entotheonella sp. in the mesohyl of the marine sponge Discodermia sp.

The lithistid sponge Discodermia dissoluta (family Theonellidae), is found in deep-waters throughout the Caribbean sea and is the source of discodermolide, a natural product with potential anticancer properties, and other secondary metabolites. As with other sponges, large numbers of microbes are harbored in the sponge mesohyl. The microbial population of the sponge mesohyl shows an abundance of large filamentous microbes. Fractionation of the dissociated sponge allowed enrichment of this microbe, which was then identified by analysis of the 16S rRNA genes. Its identity was confirmed through the use of fluorescent in situ hybridization. These studies have allowed the identification of this eubacterial microbe as belonging to the genus Entotheonella.

The diversity of the bacterial communities associated with the azooxanthellate hexacoral Cirrhipathes lutkeni.

This study examined the symbiotic microbiota of the hexacoral Cirrhipathes lutkeni using traditional plate culture, fluorescence in situ hybridization (FISH) and 16S rDNA characterization. FISH counts for the whole coral (holobiont) showed a major presence of gamma-Proteobacteria (22%) and Actinobacteria (19%), followed by alpha-Proteobacteria (14%), Firmicutes (9%), Cytophaga-Flavobacterium (7%), beta-Proteobacteria (6%) and Chloroflexi (2%). In contrast to the diversity observed by FISH, plate cultures were found to be selective for gamma-Proteobacteria (22 cultures) with the exception of an Actinobacterium. The methods employed in this study detected 76% of all microbes estimated by DAPI staining of C. lutkeni homogenates. The absence of zooxanthellae in this particular hexacoral was confirmed by PCR and spectrophotometry using fresh tissue isolated from the holobiont. This is the first study describing the microbial associations of shallow-water hexacorallia, which opens further insight into coral microbial ecology and may enhance the search for novel natural products in the near future.

Use of batch culture and a two-stage continuous culture system to study the effect of supplemental alpha-lactalbumin and glycomacropeptide on mixed populations of human gut bacteria.

Certain milk factors can promote the growth of a host-friendly gastrointestinal microflora. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts. The effect of formula supplementation with two such factors was investigated in this study. Infant faecal specimens were used to ferment formulas supplemented with glycomacropeptide and alpha-lactalbumin in a two-stage compound continuous culture model. Bacteriology was determined by fluorescence in situ hybridisation. Vessels that contained breast milk as well as alpha-lactalbumin and glycomacropeptide had stable counts of bifidobacteria while lactobacilli increased significantly only in vessels with breast milk. Bacteroides, clostridia and Escherichia coli decreased significantly in all runs. Acetate was the principal acid found along with high amounts of propionate and lactate. Supplementation of infant formulas with appropriate milk proteins may be useful in simulating the beneficial bacteriological effects of breast milk.

rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli.

OBJECTIVES: Certain milk factors may help to promote the growth of a host-friendly colonic microflora (e.g. bifidobacteria, lactobacilli) and explain why breast-fed infants experience fewer and milder intestinal infections than those who are formula-fed. The effects of supplementation of formula with two such milk factors was investigated in this study. MATERIALS AND METHODS: Infant rhesus macaques were breast-fed, fed control formula, or formula supplemented with glycomacropeptide (GMP) or alpha-lactalbumin (alpha-LA) from birth to 5 months of age. Blood was drawn monthly and rectal swabs were collected weekly. At 4.5 months of age, 10(8) colony-forming units of enteropathogenic E.coli O127, strain 2349/68 (EPEC) was given orally and the response to infection assessed. The bacteriology of rectal swabs pre- and post-infection was determined by culture independent fluorescence in situ hybridization. RESULTS: Post-challenge, breast-fed infants and infants fed alpha-LA-supplemented formula had no diarrhea, whilst those infants fed GMP-supplemented formula had intermittent diarrhea. In infants fed control formula the diarrhea was acute. CONCLUSIONS: Supplementation of infant formula with appropriate milk proteins may be useful for improving the infant's ability to resist acute infection caused by E.coli.