Sialic acid-based probiotic intervention in lactating mothers improves the neonatal gut microbiota and immune responses by regulating sialylated milk oligosaccharide synthesis via the gut-breast axis.

Human milk oligosaccharides (HMOs) are vital milk carbohydrates that help promote the microbiota-dependent growth and immunity of infants. Sialic acid (SA) is a crucial component of sialylated milk oligosaccharides (S-MOs); however, the effects of SA supplementation in lactating mothers on S-MO biosynthesis and their breastfed infants are unknown. Probiotic intervention during pregnancy or lactation demonstrates promise for modulating the milk glycobiome. Here, we evaluated whether SA and a probiotic (Pro) mixture could increase S-MO synthesis in lactating mothers and promote the microbiota development of their breastfed neonates. The results showed that SA+Pro intervention modulated the gut microbiota and 6'-SL contents in milk of maternal rats more than the SA intervention, which promoted Lactobacillus reuteri colonization in neonates and immune development. Deficient 6'-SL in the maternal rat milk of St6gal1 knockouts (St6gal1-/-) disturbed intestinal microbial structures in their offspring, thereby impeding immune tolerance development. SA+Pro intervention in lactating St6gal1± rats compromised the allergic responses of neonates by promoting 6'-SL synthesis and the neonatal gut microbiota. Our findings from human mammary epithelial cells (MCF-10A) indicated that the GPR41-PI3K-Akt-PPAR pathway helped regulate 6'-SL synthesis in mammary glands after SA+Pro intervention through the gut - breast axis. We further validated our findings using a human-cohort study, confirming that providing SA+Pro to lactating Chinese mothers increased S-MO contents in their breast milk and promoted gut Bifidobacterium spp. and Lactobacillus spp. colonization in infants, which may help enhance immune responses. Collectively, our findings may help alter the routine supplementation practices of lactating mothers to modulate milk HMOs and promote the development of early-life gut microbiota and immunity.

Multispecies probiotics complex improves bile acids and gut microbiota metabolism status in an in vitro fermentation model.

The consumption of probiotics has been extensively employed for the management or prevention of gastrointestinal disorders by modifying the gut microbiota and changing metabolites. Nevertheless, the probiotic-mediated regulation of host metabolism through the metabolism of bile acids (BAs) remains inadequately comprehended. The gut-liver axis has received more attention in recent years due to its association with BA metabolism. The objective of this research was to examine the changes in BAs and gut microbiota using an in vitro fermentation model. The metabolism and regulation of gut microbiota by commercial probiotics complex containing various species such as Lactobacillus, Bifidobacterium, and Streptococcus were investigated. The findings indicated that the probiotic strains had produced diverse metabolic profiles of BAs. The probiotics mixture demonstrated the greatest capacity for Bile salt hydrolase (BSH) deconjugation and 7α-dehydroxylation, leading to a significant elevation in the concentrations of Chenodeoxycholic acid, Deoxycholic acidcholic acid, and hyocholic acid in humans. In addition, the probiotic mixtures have the potential to regulate the microbiome of the human intestines, resulting in a reduction of isobutyric acid, isovaleric acid, hydrogen sulfide, and ammonia. The probiotics complex intervention group showed a significant increase in the quantities of Lactobacillus and Bifidobacterium strains, in comparison to the control group. Hence, the use of probiotics complex to alter gut bacteria and enhance the conversion of BAs could be a promising approach to mitigate metabolic disorders in individuals.

Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method.

The identification and quantification of viable bacteria at the species/strain level in compound probiotic products is challenging now. Molecular biology methods, e.g., propidium monoazide (PMA) combination with qPCR, have gained prominence for targeted viable cell counts. This study endeavors to establish a robust PMA-qPCR method for viable Lacticaseibacillus rhamnosus detection and systematically validated key metrics encompassing relative trueness, accuracy, limit of quantification, linear, and range. The inclusivity and exclusivity notably underscored high specificity of the primers for L. rhamnosus, which allowed accurate identification of the target bacteria. Furthermore, the conditions employed for PMA treatment were fully verified by 24 different L. rhamnosus including type strain, commercial strains, etc., confirming its effective discrimination between live and dead bacteria. A standard curve constructed by type strain could apply to commercial strains to convert qPCR Cq values to viable cell numbers. The established PMA-qPCR method was applied to 46 samples including pure cultures, probiotics as food ingredients, and compound probiotic products. Noteworthy is the congruity observed between measured and theoretical values within a 95% confidence interval of the upper and lower limits of agreement, demonstrating the relative trueness of this method. Moreover, accurate results were obtained when viable L. rhamnosus ranging from 103 to 108 CFU/mL. The comprehensive appraisal of PMA-qPCR performances provides potential industrial applications of this new technology in quality control and supervision of probiotic products.

Exploring the mechanism of probiotics in enhancing the utilization of chemical components (or polyphenols) of grape seed extract.

Fecal samples from 20 healthy adults were collected for in vitro fermentation experiments to investigate the effects of combined probiotics on the utilization of grape seed extract in humans. After fermenting for 24 h, short-chain fatty acids, metabolites, and gut microbiota composition were analyzed. Short-chain fatty acids in the grape seed extract probiotics group were significantly higher than those in the grape seed extract group. Probiotics significantly enhanced the conversion and utilization of catechins and epicatechins in grape seed extract group and increased the production of 3-hydroxyphenylacetic acid. The 16S rRNA sequencing results revealed that compound probiotics significantly increased the relative abundance of Lacticaseibacillus, HT002, Bifidobacterium, and Lactobacillus and reduced that of Escherichia-Shigella. Our findings showed considerable individual variability in the metabolic utilization of grape seed extract in humans. The consumption of probiotics appears to significantly enhance the utilization.

Effect of synbiotic supplementation on immune parameters and gut microbiota in healthy adults: a double-blind randomized controlled trial.

Synbiotics are increasingly used by the general population to boost immunity. However, there is limited evidence concerning the immunomodulatory effects of synbiotics in healthy individuals. Therefore, we conducted a double-blind, randomized, placebo-controlled study in 106 healthy adults. Participants were randomly assigned to receive either synbiotics (containing Bifidobacterium lactis HN019 1.5 × 108 CFU/d, Lactobacillus rhamnosus HN001 7.5 × 107 CFU/d, and fructooligosaccharide 500 mg/d) or placebo for 8 weeks. Immune parameters and gut microbiota composition were measured at baseline, mid, and end of the study. Compared to the placebo group, participants receiving synbiotic supplementation exhibited greater reductions in plasma C-reactive protein (P = 0.088) and interferon-gamma (P = 0.008), along with larger increases in plasma interleukin (IL)-10 (P = 0.008) and stool secretory IgA (sIgA) (P = 0.014). Additionally, synbiotic supplementation led to an enrichment of beneficial bacteria (Clostridium_sensu_stricto_1, Lactobacillus, Bifidobacterium, and Collinsella) and several functional pathways related to amino acids and short-chain fatty acids biosynthesis, whereas reduced potential pro-inflammatory Parabacteroides compared to baseline. Importantly, alternations in anti-inflammatory markers (IL-10 and sIgA) were significantly correlated with microbial variations triggered by synbiotic supplementation. Stratification of participants into two enterotypes based on pre-treatment Prevotella-to-Bacteroides (P/B) ratio revealed a more favorable effect of synbiotic supplements in individuals with a higher P/B ratio. In conclusion, this study suggested the beneficial effects of synbiotic supplementation on immune parameters, which were correlated with synbiotics-induced microbial changes and modified by microbial enterotypes. These findings provided direct evidence supporting the personalized supplementation of synbiotics for immunomodulation.

Gut microbiota decreased inflammation induced by chronic unpredictable mild stress through affecting NLRP3 inflammasome.

Dysbiosis of the gut microbiota is associated with the development of depression, but the underlying mechanism remains unclear. The aim of this study was to determine the relationship between microbiota and NLRP3 inflammasome induced by chronic unpredictable mild stress (CUMS). Fecal transplantation (FMT) experiment was conducted to elucidate the potential mechanism. Levels of NLRP3 inflammasome, microbiota, inflammatory factors and tight junction proteins were measured. CUMS stimulation significantly increased the levels of NLRP3, Caspase-1 and ASC in brain and colon(p<0.05), decreased the levels of tight junction proteins Occludin and ZO-1 (p<0.05). Interestingly, increased NLRP3 inflammasome and inflammatory cytokines and decreased tight junction proteins were found in antibiotic-treated (Abx) rats received CUMS rat fecal microbiota transplantation. Furthermore, fecal microbiota transplantation altered the microbiota in Abx rats, which partially overlapped with that of the donor rats. Importantly, probiotic administration amended the alteration of microbiota induced by CUMS treatment, then reduced the levels of NLRP3 inflammasome and inflammatory factors. In conclusion, these findings suggested that depression-like behaviors induced by CUMS stimulation were related to altered gut microbiota, broke the intestinal barrier, promoted the expression of NLRP3 inflammasome and elevated inflammation. Therefore, improving the composition of microbiota via probiotic can attenuate inflammation by amending the microbiota and suppressing the activation of NLRP3 inflammasome, which is considered as a novel therapeutic strategy for depression.

Dietary macrominerals: Updated review of their role and orchestration in human nutrition throughout the life cycle with sex differences.

Macrominerals play vital roles in a multitude of physiologic systems. A myriad of biochemical reactions are dependent on or affected by these electrolytes. The current review attempts to identify the role of macrominerals as calcium, phosphorus, magnesium, sodium, potassium and sulfur in human health, in addition to their absorption and homeostasis inside the body. We also focused on their amount in major food sources and the recommended daily intake of each macromineral. In addition, a deep insight into the orchestration of the 6 different macrominerals' requirements is presented across the human life cycle, beginning from fertility and pregnancy, and reaching adulthood and senility, with insight on interactions among them and underlying action mechanisms. The effect of sex is also presented for each mineral at each life stage to highlight the different daily requirements and/ or effects. The current review identified the role of macrominerals in human health, in addition to their absorption and homeostasis in the body. Based on the in-depth understanding of the factors influencing the metabolism of macrominerals, we could better explore their safety and possible therapeutic potential in specific disorders. There is still a need to precisely demonstrate the bioavailability of macrominerals from various types of functional food.

Current and potential trends in the bioactive properties and health benefits of Prunus mume Sieb. Et Zucc: a comprehensive review for value maximization.

Prunus mume Sieb. Et Zucc (P. mume) is an acidic fruit native to China (named Chinese Mei or greengage plum). It is currently cultivated in several Asian countries, including Japan ("Ume"), Korea (Maesil), and Vietnam (Mai or Mo). Due to its myriad nutritional and functional properties, it is accepted in different countries, and its characteristics account for its commercialization. In this review, we summarize the information on the bioactive compounds from the fruit of P. mume and their structure-activity relationships (SAR); the pulp has the highest enrichment of bioactive chemicals. The nutritional properties of P. mume and the numerous uses of its by-products make it a potential functional food. P. mume extracts exhibit antioxidant, anticancer, antimicrobial, and anti-hyperuricaemic properties, cardiovascular protective effects, and hormone regulatory properties in various in vitro and in vivo assays. SAR shows that the water solubility, molecular weight, and chemical conformation of P. mume extracts are closely related to their biological activity. However, further studies are needed to evaluate the fruit's potential nutritional and functional therapeutic mechanisms. The industrial process of large-scale production of P. mume and its extracts as functional foods or nutraceuticals needs to be further optimized.

Isomaltooligosaccharides Sustain the Growth of Prevotella Both In Vitro and in Animal Models.

The human digestive tract is colonized by trillions of bacterial cells that play important roles in human health and diseases. It is well known that dietary habits are associated with human microbiota enterotypes. However, the factors that determine the enterotype still remain elusive. In this study, it was first examined, via in vitro batch fermentation, how different carbohydrates affect the Bacteroides and Prevotella enterotypes. Among the 11 substrates (fructo-, galacto-, xylo-, manno-, and isomalto-oligosaccharides [IMO] and lactulose, raffinose, starch, inulin [INU], mannitol, and xylitol) tested, IMO, INU, and starch were found to sustain the growth of Prevotella through batch fermentation. The development of the Prevotella and Bacteroides enterotypes was further simulated in chemostats using fecal samples. IMO coupled with faster dilution rates and lower pH were required to sustain the growth of Prevotella copri in the chemostat based on 16S rRNA gene and metagenomic sequencing. Meanwhile, starch with relatively lower dilution rates and higher pH was required to support the development of the Bacteroides enterotype. Amylo-α-1,6-glucosidase, pectin, and xylan lyases were the carbohydrate-active enzymes associated with the Prevotella enterotype. The Bacteroides enterotype was associated with more diversified carbohydrate-active enzymes. Consistently, since honey contains high isomaltose content, mice fed IMO and honey displayed an increased relative abundance of Prevotella in the colon. In conclusion, both in vitro systems and a mouse model were used to demonstrate that IMO maintains the Prevotella enterotype. This result provides insight into the nutritional requirements underlying gut enterotype formation. IMPORTANCE The Prevotella enterotype type is a human traditional enterotype with high dietary fiber intake, which is related to healthy ageing and Parkinson's disease development. Manipulations of the dwelled gut microbes by dietary isomalto-oligosaccharides efficiently sustained Prevotella type enterotypes, indicating that it can be used in the improvement of elderly health by increasing the gut transit time.

Potential Health-Promoting Effects of Two Candidate Probiotics Isolated from Infant Feces Using an Immune-Based Screening Strategy.

Commensal microorganisms in the human gut are a good source of candidate probiotics, particularly those with immunomodulatory effects that may improve health outcomes by regulating interactions between the gut microbiome and distal organs. Previously, we used an immune-based screening strategy to select two potential probiotic strains from infant feces in China, Bifidobacterium breve 207-1 (207-1) and Lacticaseibacillus paracasei 207-27 (207-27). In this study, the in vitro immunological effects and potential in vivo general health benefits of these two strains were evaluated using Lacticaseibacillus rhamnosus GG (LGG) as the control. The results showed that 207-1 and 207-27 significantly and differentially modulated the cytokine profiles of primary splenic cells, while did not induce abnormal systemic immune responses in healthy mice. They also modulated the gut microbiota composition in a strain-dependent manner, thus decreasing Gram-negative bacteria and increasing health-promoting taxa and short-chain fatty acid levels, particularly butyric acid. Conclusively, 207-1 and 207-27 shaped a robust gut environment in healthy mice in a strain-specific manner. Their potential immunomodulatory effects and other elite properties will be further explored using animal models of disease and subsequent clinical trials. This immune-based screening strategy is promising in efficiently and economically identifying elite candidate probiotics.

Combined probiotics attenuate chronic unpredictable mild stress-induced depressive-like and anxiety-like behaviors in rats.

Increasing evidence indicated that probiotics can be effective in improving behaviors similar to depression and anxiety disorders. However, the underlying mechanisms remain unclear, as is the effects of single vs. combined probiotics on depression and anxiety. This study aimed to determine whether combined probiotics could attenuate depressive-like and anxiety-like behavior induced by chronic unpredictable mild stress (CUMS) and its potential mechanisms. Rats underwent CUMS treatment and then administered Lactobacillus rhamnosus HN001 (HN001) or Bifidobacterium animalis subsp. lactis HN019 (HN019), alone or in combination. Levels of neurotransmitters, inflammatory factors, and the gut microbiota were measured. HN001 and (or) HN019 treatment improved depressive-like and anxiety-like behavior in rats, including increased moving distance and exploratory behavior (p < 0.05). In addition, altered gut microbiota structure induced by CUMS was amended by HN001 and/or HN019 (p < 0.05). HN001 and/or HN019 intervention also remarkably normalized levels of 5-HT, DA, NE, HVA, DOPAC, HIAA, TNF-α, IL-6, IL-18 and IL-1ß in CUMS rats (p < 0.05). Furthermore, the effects of combined probiotics on decreasing inflammation and improved gut microbiota (Chao1 index and ACE index, p < 0.05) were superior to the single probiotics. Moreover, spearman analysis showed a certain correlation between the different microbiota, such as Firmicutes, Bacteroidetes, Verrucomicrobias, Proteobacterias and Actinobacterias, and inflammation and neurotransmitters. These findings suggested that CUMS induced depressive and anxiety-like behaviors can be alleviated by the combination of probiotics, which was possibly associated with the alterations in the gut microbiota composition and increased neurotransmitters and decreased inflammatory factors.

Studies and Application of Sialylated Milk Components on Regulating Neonatal Gut Microbiota and Health.

Breast milk is rich in sialic acids (SA), which are commonly combined with milk oligosaccharides and glycoconjugates. As a functional nutrient component, SA-containing milk components have received increasing attention in recent years. Sialylated human milk oligosaccharides (HMOs) have been demonstrated to promote the growth and metabolism of beneficial gut microbiota in infants, bringing positive outcomes to intestinal health and immune function. They also exhibit antiviral and bacteriostatic activities in the intestinal mucosa of new-borns, thereby inhibiting the adhesion of pathogens to host cells. These properties play a pivotal role in regulating the intestinal microbial ecosystem and preventing the occurrence of neonatal inflammatory diseases. In addition, some recent studies also support the promoting effects of sialylated HMOs on neonatal bone and brain development. In addition to HMOs, sialylated glycoproteins and glycolipids are abundant in milk, and are also critical to neonatal health. This article reviews the current research progress in the regulation of sialylated milk oligosaccharides and glycoconjugates on neonatal gut microbiota and health.

Lactobacilli and bifidobacteria derived from infant intestines may activate macrophages and lead to different IL-10 secretion.

In this study, three strains of lactobacilli and bifidobacteria originally isolated from healthy infants, were tested for their abilities to activate RAW264.7 cells. Gene expression and cytokine production of interleukin-10 (IL-10) of RAW264.7 cells were evaluated. The activation of extracellular regulated protein kinases 1/2 (ERK1/2), p38, and nuclear factor-κB (NK-κB) were also assessed. These results suggest lactobacilli and bifidobacteria in infants may promote production of IL-10 in macrophages, conferring a protective effect in hosts suffering from inflammation. Dimerization of TLR2 and MyD88 and subsequent phosphorylation of the key downstream signaling molecules, such as MAPKs and NK-κB, may be one of the key underlying mechanisms of activation of macrophages by these microbes. Bifidobacteria and lactobacilli induced macrophages to secrete IL-10 in a different manner, which may relate to their abilities to activate key signaling pathways mediated by TLR2 and MyD88.

Nondigestible Carbohydrates Affect Metabolic Health and Gut Microbiota in Overweight Adults after Weight Loss.

BACKGROUND: The composition of diets consumed following weight loss (WL) can have a significant impact on satiety and metabolic health. OBJECTIVE: This study was designed to test the effects of including a nondigestible carbohydrate to achieve weight maintenance (WM) following a period of WL. METHODS: Nineteen volunteers [11 females and 8 males, aged 20-62 y; BMI (kg/m2): 27-42] consumed a 3-d maintenance diet (15%:30%:55%), followed by a 21-d WL diet (WL; 30%:30%:40%), followed by 2 randomized 10-d WM diets (20%:30%:50% of energy from protein:fat:carbohydrate) containing either resistant starch type 3 (RS-WM; 22 or 26 g/d for females and males, respectively) or no RS (C-WM) in a within-subject crossover design without washout periods. The primary outcome, WM after WL, was analyzed by body weight. Secondary outcomes of fecal microbiota composition and microbial metabolite concentrations and gut hormones were analyzed in fecal samples and blood plasma, respectively. All outcomes were assessed at the end of each dietary period. RESULTS: Body weight was similar after the RS-WM and C-WM diets (90.7 and 90.8 kg, respectively), with no difference in subjectively rated appetite. During the WL diet period plasma ghrelin increased by 36% (P < 0.001), glucose-dependent insulinotropic polypeptide (GIP) decreased by 33% (P < 0.001), and insulin decreased by 46% (P < 0.001), but no significant differences were observed during the RS-WM and C-WM diet periods. Fasting blood glucose was lower after the RS-WM diet (5.59 ± 0.31 mmol/L) than after the C-WM diet [5.75 ± 0.49 mmol/L; P = 0.015; standard error of the difference between the means (SED): 0.09]. Dietary treatments influenced the fecal microbiota composition (R2 = 0.054, P = 0.031) but not diversity. CONCLUSIONS: The metabolic benefits, for overweight adults, from WL were maintained through a subsequent WM diet with higher total carbohydrate intake. Inclusion of resistant starch in the WM diet altered gut microbiota composition positively and resulted in lower fasting glucose compared with the control, with no apparent change in appetite. This trial was registered at clinicaltrials.gov as NCT01724411.

Osteopontin Levels in Human Milk Vary Across Countries and Within Lactation Period: Data From a Multicenter Study.

OBJECTIVES: Osteopontin (OPN) is a multifunctional protein expressed in many cell types, tissues and body fluids with the highest concentrations found in milk; significantly higher in human than in bovine milk. Intervention studies have indicated beneficial effects of supplementing infant formula with bovine OPN. In this multicenter study, we determined the OPN content in human milk samples from 629 Chinese, Danish, Japanese and Korean mothers. METHODS: At each study site, milk samples were collected and analyzed for OPN and protein concentration using ELISA and infrared spectroscopy, respectively. RESULTS: A total of 829 milk samples from 629 women were included. When delivering the first sample, mean maternal age was 31.4 years (SD 4.0), and median infant age was 13.4 weeks (interquartile range 4.6-17.9). The median OPN concentration varied across sites; from 99.7 mg/L in Danish, 185.0 mg/L in Japanese, 216.2 mg/L in Korean to 266.2 mg/L in Chinese mothers (P < 0.001), corresponding to 1.3%, 2.4%, 1.8% and 2.7% of the total protein content (OPN/protein%) (P < 0.05), respectively. Based on 75 Chinese and 33 Japanese mothers delivering more than 1 sample, multilevel (mixed model) linear regression analysis showed a decrease in OPN concentration with infant age (ß = (-11.3), 95% confidence interval (CI) = (-13.9) to (-8.8) and ß = (-2.1), 95% CI = (-3.2) to (-0.9), respectively). CONCLUSIONS: In this large multicenter study, we observed statistically significant differences in the OPN concentration and the OPN/protein% in human milk samples between countries. Based on mothers delivering more than 1 sample, a significant decrease within the lactation period was observed.

Fucosylated Human Milk Oligosaccharides and N-Glycans in the Milk of Chinese Mothers Regulate the Gut Microbiome of Their Breast-Fed Infants during Different Lactation Stages.

The milk glycobiome has a significant impact on the gut microbiota of infants, which plays a pivotal role in health and development. Fucosylated human milk oligosaccharides (HMOs) and N-glycans on milk proteins are beneficial for the development of healthy gut microbiota, and the fucosylation levels of these glycans can be affected by the maternal fucosyltransferase 2 gene (FUT2). Here, we present results of longitudinal research on paired milk and stool samples from 56 Chinese mothers (CMs) and their breast-fed children. Changes of HMOs and fucosylated N-glycans in milk of CMs at different lactation stages were detected, which allowed characterization of the major differences in milk glycans and consequential effects on the gut microbiome of infants according to maternal FUT2 status. Significant differences in the abundance of total and fucosylated HMOs between secretor and nonsecretor CMs were noted, especially during early lactation. Despite a tendency toward decreasing milk protein concentrations, the fucosylation levels of milk N-glycans increased during late lactation. The changes in the levels of fucosylated HMOs and milk N-glycans were highly correlated with the growth of Bifidobacterium spp. and Lactobacillus spp. in the gut of infants during early and later lactation, respectively. Enriched expression of genes encoding glycoside hydrolases, glycosyl transferases, ATP-binding cassette (ABC) transporters, and permeases in infants fed by secretor CMs contributed to the promotion of these bacteria in infants. Our data highlight the important role of fucosylated milk glycans in shaping the gut microbiome of infants and provide a solid foundation for development of "personalized" nutrition for Chinese infants. IMPORTANCE Human milk glycans provide a broad range of carbon sources for gut microbes in infants. Levels of protein glycosylation in human milk vary during lactation and may also be affected by the stages of gestation and lactation and by the secretor status of the mother. This was the first study to evaluate systematically dynamic changes in human milk oligosaccharides and fucosylated N-glycans in the milk of Chinese mothers with different secretor statuses during 6 months of lactation. Given the unique single nucleotide polymorphism site (rs1047781, A385T) on the fucosyltransferase 2 gene among Chinese populations, our report provides a specific insight into the milk glycobiome of Chinese mothers, which may exert effects on the gut microbiota of infants that differ from findings from other study cohorts.

Effect of probiotics on digestibility and immunity in infants: A study protocol for a randomized controlled trial.

The gastrointestinal (GI) tract of a fetus in utero is sterile but it becomes colonized with environmental microorganisms shortly after birth. Since the gut microbiota undergoes substantial changes in early life, healthy gut microflora is essential to an infant's gut health and immune system and probably also has an effect on overall health status in later life. Probiotics, defined as viable microbial preparations that have a beneficial effect on the health of the host, represent a rapidly expanding field. Although randomized controlled trials using probiotics in infants have shown promising results in the prevention and treatment of common diseases such as diarrhea and allergy, little is known about whether probiotics could offer benefits to healthy infants. We have designed a randomized controlled trial to test the hypothesis that an oral preparation of probiotics is superior to placebo in improving digestive and immune function in healthy infants.The trial will be a randomized, double-blind, placebo-controlled, 2-parallel-group study in Shanghai, China. After a 2-week run-in period, 200 exclusively formula-fed healthy infants aged 4 to 6 months will be randomly allocated to receive either a probiotic product containing Bifidobacterium infantis R0033, Bifidobacterium bifidum R0071, and Lactobacillus helveticus R0052 or an identical placebo once daily for 4 weeks and will be followed up for 8 weeks. The duration of the subject's participation will be 14 weeks, with a total of 5 visits: inclusion (Visit 1, Day 1), start of intervention (V2, D15), end of intervention (V3, D44), and follow-up (V4 and V5, D72 and D100). Stool and saliva samples will be collected at the first 3 visits to measure microbial populations and secretory immunoglobulin A (SIgA), respectively. Physical examination will be performed at each visit, and tolerance records will be completed 1 day prior to each visit. The primary endpoints will be the changes in the composition of fecal microbiota, particularly the Bifidobacterium bifidum population. The secondary endpoints will include the change in salivary SIgA level, growth parameters, digestive tolerance, and adverse events.An effective, practical, and acceptable probiotic intervention in manipulating the gut microbiota and boosting the immune system in formula-fed infants would represent a major clinical advance. The administration of probiotic supplementation or follow-on formula to infant may be associated with some clinic benefits.

Unique Organization of Extracellular Amylases into Amylosomes in the Resistant Starch-Utilizing Human Colonic Firmicutes Bacterium Ruminococcus bromii.

UNLABELLED: Ruminococcus bromii is a dominant member of the human gut microbiota that plays a key role in releasing energy from dietary starches that escape digestion by host enzymes via its exceptional activity against particulate "resistant" starches. Genomic analysis of R. bromii shows that it is highly specialized, with 15 of its 21 glycoside hydrolases belonging to one family (GH13). We found that amylase activity in R. bromii is expressed constitutively, with the activity seen during growth with fructose as an energy source being similar to that seen with starch as an energy source. Six GH13 amylases that carry signal peptides were detected by proteomic analysis in R. bromii cultures. Four of these enzymes are among 26 R. bromii proteins predicted to carry dockerin modules, with one, Amy4, also carrying a cohesin module. Since cohesin-dockerin interactions are known to mediate the formation of protein complexes in cellulolytic ruminococci, the binding interactions of four cohesins and 11 dockerins from R. bromii were investigated after overexpressing them as recombinant fusion proteins. Dockerins possessed by the enzymes Amy4 and Amy9 are predicted to bind a cohesin present in protein scaffoldin 2 (Sca2), which resembles the ScaE cell wall-anchoring protein of a cellulolytic relative, R. flavefaciens. Further complexes are predicted between the dockerin-carrying amylases Amy4, Amy9, Amy10, and Amy12 and two other cohesin-carrying proteins, while Amy4 has the ability to autoaggregate, as its dockerin can recognize its own cohesin. This organization of starch-degrading enzymes is unprecedented and provides the first example of cohesin-dockerin interactions being involved in an amylolytic system, which we refer to as an "amylosome." IMPORTANCE: Fermentation of dietary nondigestible carbohydrates by the human colonic microbiota supplies much of the energy that supports microbial growth in the intestine. This activity has important consequences for health via modulation of microbiota composition and the physiological and nutritional effects of microbial metabolites, including the supply of energy to the host from short-chain fatty acids. Recent evidence indicates that certain human colonic bacteria play keystone roles in degrading nondigestible substrates, with the dominant but little-studied species Ruminococcus bromii displaying an exceptional ability to degrade dietary resistant starches (i.e., dietary starches that escape digestion by host enzymes in the upper gastrointestinal tract because of protection provided by other polymers, particle structure, retrogradation, or chemical cross-linking). In this report, we reveal the unique organization of the amylolytic enzyme system of R. bromii that involves cohesin-dockerin interactions between component proteins. While dockerins and cohesins are fundamental to the organization of cellulosomal enzyme systems of cellulolytic ruminococci, their contribution to organization of amylases has not previously been recognized and may help to explain the starch-degrading abilities of R. bromii.

Some are more equal than others: the role of "keystone" species in the degradation of recalcitrant substrates.

The microbial communities found in the mammalian large intestine and rumen efficiently degrade many recalcitrant substrates that are resistant to the host's digestive enzymes. These communities are known from molecular profiling to be highly diverse at the species and strain level, but it may be that only certain specialized organisms ("keystone species") have the ability to initiate degradation of such substrates, thus releasing energy on which the rest of the community depends. We have recently reported that Ruminococcus bromii has a superior ability to degrade certain forms of particulate resistant starch (RS) when compared with other highly abundant species of amylolytic bacteria found in the human colon and have presented evidence that this bacterium provides an example of a keystone species within the microbial community with respect to RS fermentation. The concept of keystone species can be equally relevant to other activities, e.g., those involved in stabilizing the community.

Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon.

The release of energy from particulate substrates such as dietary fiber and resistant starch (RS) in the human colon may depend on the presence of specialist primary degraders (or 'keystone species') within the microbial community. We have explored the roles of four dominant amylolytic bacteria found in the human colon in the degradation and utilization of resistant starches. Eubacterium rectale and Bacteroides thetaiotaomicron showed limited ability to utilize RS2- and RS3-resistant starches by comparison with Bifidobacterium adolescentis and Ruminococcus bromii. In co-culture, however, R. bromii proved unique in stimulating RS2 and RS3 utilization by the other three bacterial species, even in a medium that does not permit growth of R. bromii itself. Having previously demonstrated low RS3 fermentation in vivo in two individuals with undetectable populations of R. bromii-related bacteria, we show here that supplementation of mixed fecal bacteria from one of these volunteers with R. bromii, but not with the other three species, greatly enhanced the extent of RS3 fermentation in vitro. This argues strongly that R. bromii has a pivotal role in fermentation of RS3 in the human large intestine, and that variation in the occurrence of this species and its close relatives may be a primary cause of variable energy recovery from this important component of the diet. This work also indicates that R. bromii possesses an exceptional ability to colonize and degrade starch particles when compared with previously studied amylolytic bacteria from the human colon.