Probiotic Characteristics of Lactiplantibacillus plantarum CECT 9435 and Its Survival and Competitive Properties Under Simulated Conditions of the Child Gut Microbiota.

Probiotics are valuable microorganisms effective in reducing malnutrition-related infections in children. In this work, a collection of lactobacilli strains representative of traditional Andean fermented beverages was in vitro screened for their capability to survive the gastrointestinal transit, to adhere to the intestinal epithelium and to compete under simulated conditions of the child gut microbiota. The results allowed the selection of the riboflavin overproducing strain Lactiplantibacillus plantarum CECT 9435 based on its good rate of survival under in vitro gastrointestinal conditions when included in a food matrix representing the fortified food supplement Incaparina. The strain also showed good adhesion to HT29 cells producing mucus and outstanding performance in E. coli competition for the adhesion to this epithelial cell line. L. plantarum CECT 9435 gut performance was also evaluated in the child intestinal microbiota simulated in a dynamic gut model (BFBL simulator). The viability of the probiotic candidate in the gut conditions was high during the 7-day intervention period, reaching over 1 × 107 counts in each of the reactors simulating the three colonic regions. The transient viability of L. plantarum CECT 9435 within the child gut microbiota and its adhesion capacity to intestinal cells could facilitate the strain potential benefits as probiotic added to fortified supplementary foods destined to malnourished children.

Encapsulation of Indicaxanthin-Rich Opuntia Green Extracts by Double Emulsions for Improved Stability and Bioaccessibility.

Opuntia ficus-indica var. Colorada fruit is an important source of indicaxanthin, a betalain with antioxidant, anti-inflammatory, and neuromodulatory potential, proven in both in vitro and in vivo models. Other betalains and phenolic compounds with bioactive activities have also been identified in Colorada fruit extracts. These compounds may degrade by their exposure to different environmental factors, so in the present research, two double emulsion systems (W1/O/W2) were elaborated using Tween 20 (TW) and sodium caseinate (SC) as surfactants to encapsulate Colorada fruit pulp extracts, with the aim of enhancing their stability during storage. Encapsulation efficiencies of up to 97.3 ± 2.7%, particle sizes between 236 ± 4 and 3373 ± 64 nm, and zeta potential values of up to ∣46.2∣ ± 0.3 mV were obtained. In addition, the evaluation of the in vitro gastro-intestinal stability and bioaccessibility of the main individual bioactives was carried out by standardized INFOGEST© protocol, obtaining the highest values for the encapsulated extract bioactives in comparison with the non-encapsulated extract (control). Especially, TW double emulsion showed bioaccessibility values of up to 82.8 ± 1.5% for the main bioactives (indicaxanthin, piscidic acid, and isorhamnetin glucoxyl-rhamnosyl-pentoside 2 (IG2)), indicating a promising potential for its use as a functional natural colorant ingredient.

The food additive xylitol enhances the butyrate formation by the child gut microbiota developed in a dynamic colonic simulator.

The gut microbiota should be included in the scientific processes of risk assessment of food additives. Xylitol is a sweetener that shows low digestibility and intestinal absorption, implying that a high proportion of consumed xylitol could reach the colonic microbiota. The present study has evaluated the dose-dependent effects of xylitol intake on the composition and the metabolic activity of the child gut-microbiota. The study was conducted in a dynamic simulator of the colonic microbiota (BFBL Gut Simulator) inoculated with a child pooled faecal sample and supplemented three times per day, for 7 days, with increasing xylitol concentrations (1 g/L, 3 g/L and 5 g/L). Sequencing of 16S rRNA gene amplicons and group-specific quantitative PCR indicated a xylitol dose-response effect on the abundance of Lachnospiraceae, particularly the genera Blautia, Anaerostipes and Roseburia. The microbial changes observed with xylitol corresponded with a dose-dependant effect on the butyrate concentration that, in parallel, favoured an increase in epithelial integrity of Caco-2 cells. The study represents a detailed observation of the bacterial taxa that are the main contributors to the metabolism of xylitol by the child gut microbiota and the results could be relevant in the risk assessment re-evaluation of xylitol as a sweetener.

Anti-Inflammatory and Gut Microbiota Modulation Potentials of Flavonoids Extracted from Passiflora foetida Fruits.

This study aimed to explore the anti-inflammatory and gut microbiota modulation potentials of flavonoid-rich fraction (PFF) extracted from Passiflora foetida fruits. The results showed that PFF markedly reduced the production of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in LPS-stimulated RAW 264.7 cells. Meanwhile, PFF treatment also effectively decreased the phosphorylation levels of MAPK, PI3K/Akt, and NF-κB signaling-pathway-related proteins (ERK, JNK, p38, Akt, and p65). Moreover, PFF had an impact on microbial composition and metabolites in a four-stage dynamic simulator of human gut microbiota (BFBL gut model). Specifically, PFF exhibited the growth-promoting ability of several beneficial bacteria, including Bifidobacterium, Enterococcus, Lactobacillus, and Roseburia, and short-chain fatty acid (SCFA) generation ability in gut microbiota. In addition, spectroscopic data revealed that PFF mainly contained five flavonoid compounds, which may be bioactive compounds with anti-inflammatory and gut microbiota modulation potentials. Therefore, PFF could be utilized as a natural anti-inflammatory agent or supplement to health products.

RotaRod and acoustic startle reflex performance of two potential mouse models for Meniere's disease.

Meniere's disease (MD) is a disorder of the inner ear characterized by chronic episodes of vertigo, tinnitus, increased aural pressure, and sensorineural hearing loss. Causes of MD are unknown, but endolymphatic hydrops is a hallmark. In addition, 5%-15% of MD cases have been identified as familial. Whole-genome sequencing studies of individuals with familial MD identified DTNA and FAM136A as candidate genes for autosomal dominant inheritance of MD. Although the exact roles of these genes in MD are unknown, FAM136A encodes a mitochondrial protein, and DTNA encodes a cytoskeletal protein involved in synapse formation and maintenance, important for maintaining the blood-brain barrier. It is also associated with a particular aquaporin. We tested vestibular and auditory function in dtna and fam136a knockout (KO) mice, using RotaRod and startle reflex-based clicker tests, respectively. Three-factor analysis of variance (ANOVA) results indicated that sex, age, and genotype were significantly correlated with reduced mean latencies to fall ("latencies") for male dtna KO mice, while only age was a significant factor for fam136a KO mice. Fam136a KO mice lost their hearing months before WTs (9-11 months vs. 15-20 months). In male dtna KO mice, divergence in mean latencies compared with other genotypes was first evident at 4 months of age, with older males having an even greater decrease. Our results indicate that fam136a gene mutations generate hearing problems, while dtna gene mutations produce balance deficits. Both mouse models should help to elucidate hearing loss and balance-related symptoms associated with MD.

Decreased retronasal olfaction and taste perception in obesity are related to saliva biochemical and microbiota composition.

Understanding the individual factors that modulate flavor perception is a central issue for the development of personalized diets strategies to fight obesity. This study aimed to investigate differences in flavor perception between adults with normal weight and those with obesity, as well as some potential biological factors related to these differences. To do that, liking and flavor perception intensity were measured against retronasal olfactory (pineapple, butter, tropical and chocolate) and taste attributes (sweetness, umami and bitter) in 77 individuals grouped as normalweight or obese, according to their body mass index (BMI). Unstimulated saliva was collected from all participants and characterized in terms of salivary flow, total protein content, total antioxidant capacity, total esterase activity and bacterial composition through 16S rDNA amplicon sequencing. The results showed that participants displayed differences in flavor perception according to their BMI group. Thus, the group with obesity showed significant lower liking and intensity scores for low calorie related food aroma (pineapple and tropical), lower taste intensity scores for sweet and umami, and a higher acceptability for umami than the group with normal weight. Significant differences between BMI groups were observed for salivary biochemical variables and specific bacterial taxa, some of which were significantly correlated to flavor intensity. This work suggests for the first time the existence of an oral-brain axis that might contribute to the development or perpetuation of obesity, which opens new and interesting avenues of research.

Role of lactic acid bacteria and yeasts in sourdough fermentation during breadmaking: Evaluation of postbiotic-like components and health benefits.

Sourdough (SD) fermentation is a traditional biotechnological process used to improve the properties of baked goods. Nowadays, SD fermentation is studied for its potential health effects due to the presence of postbiotic-like components, which refer to a group of inanimate microorganisms and/or their components that confer health benefits on the host. Some postbiotic-like components reported in SD are non-viable microorganisms, short-chain fatty acids, bacteriocins, biosurfactants, secreted proteins/peptides, amino acids, flavonoids, exopolysaccharides, and other molecules. Temperature, pH, fermentation time, and the composition of lactic acid bacteria and yeasts in SD can impact the nutritional and sensory properties of bread and the postbiotic-like effect. Many in vivo studies in humans have associated the consumption of SD bread with higher satiety, lower glycemic responses, increased postprandial concentrations of short-chain fatty acids, and improvement in the symptoms of metabolic or gastrointestinal-related diseases. This review highlights the role of bacteria and yeasts used for SD, the formation of postbiotic-like components affected by SD fermentation and the baking process, and the implications of functional SD bread intake for human health. There are few studies characterizing the stability and properties of postbiotic-like components after the baking process. Therefore, further research is necessary to develop SD bread with postbiotic-related health benefits.

Immunological Activity and Gut Microbiota Modulation of Pectin from Kiwano (Cucumis metuliferus) Peels.

For developing the recycling of fruit by-products from kiwano, a polysaccharide was extracted from kiwano (Cucumis metuliferus) peels, namely Cucumis metuliferus peels polysaccharide (CMPP), with the aim of investigating the potential beneficial effects. The composition of polysaccharides was analyzed by chemical methods. RAW264.7 macrophages cells and the microbiota dynamics simulator (BFBL gut model) were used for in vitro study. The result showed that CMPP mainly consists of glucuronic acid, arabinose, galactose and rhamnose. By intervening with RAW264.7 cells, CMPP promoted cell proliferation and showed immune-enhancing activity, which significantly (p < 0.05) induced the release of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) at a concentration of 50 µg/mL. In addition, CMPP had an impact on the composition of the gut bacteria, increasing the growth of Akkermansia, Bacteroides, Bifidobacterium, Feacalibacterium, and Roseburia. During the intake period, acetic, butyric and propionic acids were all increased, especially (p < 0.05) in the descending colon. Moreover, a decrease in ammonia concentration (10.17 ± 0.50 mM in the ascending colon, 13.21 ± 1.54 mM in the transverse colon and 13.62 ± 0.45 mM in the descending colon, respectively) was observed. In summary, CMPP can be considered as a pectin, showed immunological activity and function of gut microbiota modulation. This study could be the scientific basis of developing kiwano peels as beneficial to human health.

Taxonomic Characterization and Short-Chain Fatty Acids Production of the Obese Microbiota.

Intestinal microbiota seems to play a key role in obesity. The impact of the composition and/or functionality of the obesity-associated microbiota have yet to be fully characterized. This work assessed the significance of the taxonomic composition and/or metabolic activity of obese- microbiota by massive 16S rRNA gene sequencing of the fecal microbiome of obese and normoweight individuals. The obese metabolic activity was also assessed by in vitro incubation of obese and normoweight microbiotas in nutritive mediums with different energy content. We found that the microbiome richness and diversity of the two groups did not differ significantly, except for Chao1 index, significantly higher in normoweight individuals. At phylum level, neither the abundance of Firmicutes or Bacteroidetes nor their ratio was associated with the body mass index. Besides, the relative proportions in Collinsella, Clostridium XIVa, and Catenibacterium were significantly enriched in obese participants, while Alistipes, Clostridium sensu stricto, Romboutsia, and Oscillibacter were significantly diminished. In regard to metabolic activity, short-chain fatty acids content was significant higher in obese individuals, with acetate being the most abundant followed by propionate and butyrate. Acetate and butyrate production was also higher when incubating obese microbiota in mediums mimicking diets with different energy content; interestingly, a reduced capability of propionate production was associated to the obese microbiome. In spite of the large interindividual variability, the obese phenotype seems to be defined more by the abundance and/or the absence of distinct communities of microorganism rather than by the presence of a specific population.

Probiotics, prebiotics, and synbiotics added to dairy products: Uses and applications to manage type 2 diabetes.

Diabetes mellitus type 2 (T2DM) is associated with hyperglycemia, insulin resistance, and gut dysbiosis. Probiotics and prebiotics can ameliorate T2DM through different mechanisms of action, such as reducing oxidative stress, or the inhibition of pro-inflammatory markers, among others. Multiple studies in vitro and in vivo have demonstrated the reduction of hyperglycemia, depressive behaviors, obesity, oxidative stress, and insulin resistance in diabetic patients through the consumption of dairy products, such as yogurt, fermented milk, and cheese, enriched with potential probiotic strains, prebiotic ingredients, and synbiotics (understood as a combination of both). Therefore, this review aims to provide an updated overview about the impact of dairy foods with probiotics, prebiotics, or synbiotics to prevent and manage T2DM, the mechanism of action related to the host health, and the future tendencies for developing new dairy foods. Despite the addition of probiotics, prebiotics, and synbiotics to dairy products could be highly beneficial, more evidence, especially from clinical trials, is needed to develop evidence-based T2DM prevention guidelines.

Fate of Methicillin-resistant Staphylococcus aureus (MRSA) under simulated acidic conditions of the human stomach.

A known amount (107 cfu/ml) of animal origin Methicillin-resistant Staphylococcus aureus (MRSA) ST398/t011/V and of human origin MRSA ST1/t127/IVa strains were individually inoculated into ricotta cheese and hamburger samples. The pH of each food matrix was gradually decreased from 6.0 down to 2.0 during a period of about 2 hr, under conditions simulating the mechanical digestion of the human stomach. Afterward, the MRSA strains were recovered by using a MRSA-specific plating medium. Although both strains showed a certain acidic resistance, they showed different responses at low pH values during the experiment: ST398 survived unharmed during the course of the experiments to the last stage at pH 2 where counts of 6.4 cfu/g for the hamburger and 7.5 log cfu/g for ricotta cheese assays were obtained. In contrast, the ST1 population was no longer detectable at pH 3 in the hamburger and at pH 2 in the ricotta cheese assays. To the best of our knowledge, this is the first study that investigates the ability of MRSA to overcome the acidic conditions of the human stomach and that adds new evidence that might contribute to expand the scientific knowledge on the significance of MRSA in the food safety debate.

A Systematic Review of Extreme Phenotype Strategies to Search for Rare Variants in Genetic Studies of Complex Disorders.

Exome sequencing has been commonly used to characterize rare diseases by selecting multiplex families or singletons with an extreme phenotype (EP) and searching for rare variants in coding regions. The EP strategy covers both extreme ends of a disease spectrum and it has been also used to investigate the contribution of rare variants to the heritability of complex clinical traits. We conducted a systematic review to find evidence supporting the use of EP strategies in the search for rare variants in genetic studies of complex diseases and highlight the contribution of rare variations to the genetic structure of polygenic conditions. After assessing the quality of the retrieved records, we selected 19 genetic studies considering EPs to demonstrate genetic association. All studies successfully identified several rare or de novo variants, and many novel candidate genes were also identified by selecting an EP. There is enough evidence to support that the EP approach for patients with an early onset of a disease can contribute to the identification of rare variants in candidate genes or pathways involved in complex diseases. EP patients may contribute to a better understanding of the underlying genetic architecture of common heterogeneous disorders such as tinnitus or age-related hearing loss.

Prebiotic galactooligosaccharides production from lactose and lactulose by Lactobacillus delbrueckii subsp. bulgaricus CRL450.

Galactooligosaccharides (GOS) are useful dietary ingredients recognized worldwide as prebiotics. In the present study, we evaluated the ß-galactosidase (ß-gal) activity of a panel of lactic acid bacteria (LAB) in order to select strains for the synthesis of oligosaccharides from lactose (GOS) and lactulose (OsLu) with a potential prebiotic effect. Fifteen strains out of 20 were able to grow on lactose and showed ß-gal activities between 0.03 and 2.06 U mg-1, whereas eleven were able to synthesize GOS. Lactobacillus delbrueckii subsp. bulgaricus CRL450, the strain with the highest ß-gal activity, synthesized a maximum of 41.3% GOS and 21.0% OsLu from lactose and lactulose, respectively, with ß-(1 → 6) and secondary ß-(1 → 3) linkages. When these compounds were tested without purifying, as carbon sources for the development of recognized probiotics and the producer strain, high growth was observed compared to non-prebiotic sugars like glucose and lactose. When the purified oligosaccharides were tested, the bacterial growth decreased, but the microorganisms displayed metabolic activity evidenced by the consumption of carbohydrates and the production of lactic acid. Additionally, the purified oligosaccharides demonstrated a bifidogenic effect. The obtained results support the potential of L. delbrueckii subsp. bulgaricus CRL450 for the production of the prebiotics GOS and OsLu and encourage the optimization of their synthesis for the design of new functional food ingredients.

Publisher Correction: Lyso-Gb3 modulates the gut microbiota and decreases butyrate production.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural Features of Three Hetero-Galacturonans from Passiflora foetida Fruits and Their in Vitro Immunomodulatory Effects.

Passiflora foetida is a horticultural plant and vital traditional Chinese herbal medicine. In our previous study, the characterization and immuno-enhancing effect of fruits polysaccharide 1 (PFP1), a water-eluted hetero-mannan from wild Passiflora foetida fruits, were investigated. Herein, another three salt-eluted novel polysaccharides, namely PFP2, PFP3, and PFP4, were obtained and structurally characterized. The results showed that PFP2, PFP3, and PFP4 were three structurally similar hetero-galacturonans with different molecular weights of 6.11 × 104, 4.37 × 104, and 3.48 × 105 g/mol, respectively. All three of these hetero-galacturonans are mainly composed of galacturonic acid, galactose, arabinose (75.69%, 80.39%, and 74.30%, respectively), and other monosaccharides including mannose, fucose, glucose, ribose, xylose, and glucuronic acid (24.31%, 19.61, and 25.70%, respectively), although differences in their backbone structure exist. Additionally, immunomodulatory assay indicated that the three hetero-galacturonans possess the ability to promote the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in RAW264.7 macrophages in a concentration-dependent manner (p < 0.05). Especially, PFP3 displayed a stronger enhancing effect than PFP2 and PFP4 at the minimum effective concentration. Therefore, the results suggested that the obtained three salt-eluted hetero-galacturonans, especially PFP3, could be utilized as immunomodulatory effectivity ingredients in nutritional/pharmaceutical industries.

Enrichment of damaging missense variants in genes related with axonal guidance signalling in sporadic Meniere's disease.

INTRODUCTION: Meniere's disease (MD) is a rare inner ear disorder with a significant genetic contribution defined by a core phenotype: episodic vertigo, sensorineural hearing loss and tinnitus. It has been mostly described in sporadic cases, familial cases being around 10% of the observed individuals. It is associated with an accumulation of endolymph in the inner ear, but the molecular underpinnings remain largely unknown. The main molecular pathways showing higher differentially expressed genes in the supporting cells of the inner ear are related to cochlea-vestibular innervation, cell adhesion and leucocyte extravasation. In this study, our objective is to find a burden of rare variants in genes that interact with the main signalling pathways in supporting cells of the inner ear in patients with sporadic MD. METHODS: We designed a targeted-sequencing panel including genes related with the main molecular pathways in supporting cells and sequenced 860 Spanish patients with sporadic MD. Variants with minor allele frequencies <0.1 in the gene panel were compared with three independent reference datasets. Variants were classified as loss of function, missense and synonymous. Missense variants with a combined annotation-dependent depletion score of >20 were classified as damaging missense variants. RESULTS: We have observed a significant burden of damaging missense variants in few key genes, including the NTN4 gene, associated with axon guidance signalling pathways in patients with sporadic MD. We have also identified active subnetworks having an enrichment of rare variants in sporadic MD. CONCLUSION: The burden of missense variants in the NTN4 gene suggests that axonal guidance signalling could be a novel pathway involved in sporadic MD.

Transcriptomic Analyses of Inner Ear Sensory Epithelia in Zebrafish.

Analysis of gene expression has the potential to assist in the understanding of multiple cellular processes including proliferation, cell-fate specification, senesence, and activity in both healthy and disease states. Zebrafish model has been increasingly used to understand the process of hearing and the development of the vertebrate auditory system. Within the zebrafish inner ear, there are three otolith organs, each containing a sensory macula of hair cells. The saccular macula is primarily involved in hearing, the utricular macula is primarily involved in balance and the function of the lagenar macula is not completely understood. The goal of this study is to understand the transcriptional differences in the sensory macula associated with different otolith organs with the intention of understanding the genetic mechanisms responsible for the distinct role each organ plays in sensory perception. The sensory maculae of the saccule, utricle, and lagena were dissected out of adult Et(krt4:GFP)sqet4 zebrafish expressing green fluorescent protein in hair cells for transcriptional analysis. The total RNAs of the maculae were isolated and analyzed by RNA GeneChip microarray. Several of the differentially expressed genes are known to be involved in deafness, otolith development and balance. Gene expression among these otolith organs was very well conserved with less than 10% of genes showing differential expression. Data from this study will help to elucidate which genes are involved in hearing and balance. Furthermore, the findings of this study will assist in the development of the zebrafish model for human hearing and balance disorders. Anat Rec, 303:527-543, 2020. © 2019 American Association for Anatomy.

Real-Time Detection of Riboflavin Production by Lactobacillus plantarum Strains and Tracking of Their Gastrointestinal Survival and Functionality in vitro and in vivo Using mCherry Labeling.

Some strains of lactic acid bacteria (LAB) produce riboflavin, a water-soluble vitamin of the B complex, essential for human beings. Here, we have evaluated riboflavin (B2 vitamin) production by five Lactobacillus plantarum strains isolated from chicha, a traditional maize-based fermented alcoholic beverage from north-western Argentina and their isogenic riboflavin-overproducing derivatives previously selected using roseoflavin. A direct fluorescence spectroscopic detection method to quantify riboflavin production in bacterial culture supernatants has been tested. Comparison of the efficiency for riboflavin fluorescence quantification with and without prior HPLC fractionation showed that the developed method is a rapid and easy test for selection of B2 vitamin-producing strains. In addition, it can be used for quantitative detection of the vitamin production in real time during bacterial growth. On the basis of this and previous analyses, the L. plantarum M5MA1-B2 riboflavin overproducer was selected for in vitro and in vivo studies after being fluorescently labeled by transfer of the pRCR12 plasmid, which encodes the mCherry protein. The labeling did not affect negatively the growth, the riboflavin production nor the adhesion of the strain to Caco-2 cells. Thus, L. plantarum M5MA1-B2[pRCR12] was evaluated for its survival under digestive tract stresses in the presence of microbiota in the dynamic multistage BFBL gut model and in a murine model. After exposure to both models, M5MA1-B2[pRCR12] could be recovered and detected by the pink color of the colonies. The results indicated a satisfactory resistance of the strain to gastric and intestinal stress conditions but a low colonization capability observed both in vitro and in vivo. Overall, L. plantarum M5MA1-B2 could be proposed as a probiotic strain for the development of functional foods.

Lyso-Gb3 modulates the gut microbiota and decreases butyrate production.

Fabry disease is a rare X-linked lysosomal storage disorder resulting from deficient activity of α-galactosidase A, leading to the accumulation of glycosphingolipids such as globotriaosylsphingosine (lyso-Gb3). The gastrointestinal symptoms of this disease may be disabling, and the life expectancy of affected patients is shortened by kidney and heart disease. Our hypothesis was that lyso-Gb3 may modify the gut microbiota. The impact of a clinically relevant concentration of lyso-Gb3 on mono- or multispecies bacterial biofilms were evaluated. A complex bacterial community from the simulated transverse colon microbiota was studied using quantitative PCR to estimate different bacterial group concentrations and a HPLC was used to estimate short-chain fatty acids concentrations. We found that lyso-Gb3 increased the biofilm-forming capacity of several individual bacteria, including Bacteroides fragilis and significantly increased the growth of B. fragilis in a multispecies biofilm. Lyso-Gb3 also modified the bacterial composition of the human colon microbiota suspension, increasing bacterial counts of B. fragilis, among others. Finally, lyso-Gb3 modified the formation of short-chain fatty acids, leading to a striking decrease in butyrate concentration. Lyso-Gb3 modifies the biology of gut bacteria, favoring the production of biofilms and altering the composition and short-chain fatty-acid profile of the gut microbiota.