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Made available in DSpace on 2021-06-25T10:25:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 This study investigated the effect of four citrus pectins on adhesion of the probiotic Limosilactobacillus fermentum PCC and integrity of the intestinal epithelial monolayers using the Caco-2 cell model. Adhesion of L. fermentum PCC was enhanced (from 35% to 54–73%) in the presence of pectins with high and moderate degree of esterification (DE), whereas it was reduced (to 2%) by a pectin with low DE. Transepithelial electrical resistance (TEER) was significantly increased upon exposure to L. fermentum PCC alone (by 33%) and, to a greater extent, by combinations with pectins (by 44 – 48%), suggesting synergistic action of bacteria and pectins. Increases in TEER were related to transcriptional responses of the tight junction genes, encoding claudin-4 and claudin-2 proteins. The overall results indicated that pectins, when applied in synbiotic combination with L. fermentum PCC, have potential to improve bacterial adhesion and the intestinal epithelial barrier. Department of Food Science Faculty of Science University of Copenhagen Department of Food Science UNESP - São Paulo State University Department of Experimental Biology Masaryk University Department of Food Science UNESP - São Paulo State University

Pectins are plant polysaccharides used in food industry as gelling and stabilizing agents. This study investigated the ability of pectins to improve survival of probiotic species Lactobacillus fermentum PCC, L. reuteri RC-14, L. rhamnosus LGG and L. paracasei F-19 in simulated gastric solution in relationship to their structural and physical properties. Electrostatic interactions between pectins and bacteria were evaluated by the Zeta-potential approach. Bacterial survival was assessed by flow cytometry and plate counting. L. fermentum PCC and L. reuteri RC-14 were more resistant to gastric conditions; their survival rate was further improved in the presence of five out of ten tested pectins. Additionally, two of the pectins had a positive effect on viability of the less resistant L. rhamnosus LGG and L. paracasei F-19. The beneficial effect was generally observed for the high-methoxylated pectins, indicating that substituted polygalacturonic acid in the backbone is essential for bacterial protection. Other pectin features associated with improved survival, included less negative Zeta-potential, higher molecular weight, as well as lower values of hydrodynamic sizes, viscosity and degree of branching. The study indicates that pectins have a potential to protect probiotic bacteria through the gastro-intestinal transit and identifies the features linked to their functionality.

Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production.

Mesophilic starter cultures, such as Lactococcus lactis, are exposed to NaCl stress during brining of cheeses influencing their viability. We investigated the effect of NaCl water solutions, used at concentrations of 3.4%, 8.0% and 15% (w/v), on viability of Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis biovar. diacetylactis and Lc. lactis subsp. lactis isolated from commercial dl -starter cultures. Viability was assessed by plate counts and flow cytometry. Bacterial viability decreased with increased concentration of NaCl in a strain-dependent manner (between 0.02% and 62% cfu at 15% (w/v) NaCl, compared with controls at 0.85% (w/v) NaCl), whereas no significant differences were detected between the subspecies. Viability levels determined by plate counts were generally lower than by flow cytometry, indicating the presence of viable but not culturable cells. Knowledge of salt tolerance of Lc. lactis strains would enable rational selection of starter cultures targeting production of cheeses with different NaCl content.

IntroductionDysfunctional gut microbiota is a common finding in patients with metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM). Recent clinical trials have assessed whether botanical fermented foods (BFFs) have beneficial effects on metabolic biomarkers, inflammatory markers and gut microbiota. The aim of this review is to critically evaluate all randomised controlled trials (RCTs) of BFF for evidence of impact on the outcome measures of these disease states.Methods and analysisFour electronic databases (Embase, MEDLINE, CENTRAL and Google Scholar) as well as the grey literature will be searched from inception to present without language or publication status restrictions applied. Eligible RCTs which have enrolled adult participants with T2DM, any MetS components or combinations of these components, treated prophylactically or therapeutically with any botanical fermented food intervention, compared with a control group (no intervention, placebo or active control) will be assessed. Primary outcomes are related to the target conditions, including metabolic biomarkers, inflammatory markers and gut microbiota composition/function. Using Covidence, two independent investigators will conduct title and abstract screening, followed by full-text screening to identify appropriate studies. Methodological quality of the trials will be assessed using the Cochrane risk of bias assessment tool. Findings will be summarised with a narrative synthesis of the differences between included studies. A meta-analysis will be conducted if sufficient data are obtained.Ethics and disseminationEthical approval is not required as primary data will not be collected. Results will be disseminated through peer-reviewed publication, conference presentations and press.PROSPERO registration numberCRD42018117766

Bacillus spp. are commonly used as probiotic species in the feed industry, however, their benefits need to be confirmed. This study describes a high throughput screening combined with the detailed characterization of endospore-forming bacteria with the aim to identify new Bacillus spp. strains for use as probiotic additives in pig feed. A total of 245 bacterial isolates derived from African fermented food, feces and soil were identified by 16S rRNA gene sequencing and screened for antimicrobial activity and growth in the presence of antibiotics, bile salts and at pH 4.0. Thirty-three Bacillus spp. isolates with the best characteristics were identified by gyrB and rpoB gene sequencing as B. amyloliquefaciens subsp. plantarum, B. amyloliquefaciens subsp. amyloliquefaciens, B. subtilis subsp. subtilis, B. licheniformis, B. mojavensis, B. pumilus and B. megaterium. These isolates were further investigated for their activity against the pathogenic bacteria, antibiotic susceptibility, sporulation rates, biofilm formation and production of glycosyl hydrolytic enzymes. Additionally, ten selected isolates were assessed for heat resistance of spores and the effect on porcine epithelial cells IPEC-J2. Isolates of B. amyloliquefaciens, B. subtilis and B. mojavensis, showed the best overall characteristics and, therefore, potential for usage as probiotic additives in feed. A large number of taxonomically diverse strains made it possible to reveal species and subspecies-specific trends, contributing to our understanding of the probiotic potential of Bacillus species.

Expression of virulence-related genes in Listeria monocytogenes incubated on cheese was assessed by real-time quantitative polymerase chain reaction. The objective of the study was to investigate the impact of sodium chloride concentration in cheese on transcription of virulence genes and, thereby, virulence potential of L. monocytogenes. The expression studies were performed with L. monocytogenes strains characterized by different tolerance to salt stress. Strains ATCC(®) 51779 and DSMZ 15675 were incubated on the Danish hard-cheese type Samsoe, with low (0.15% [wt/wt]) and high (3.6% [wt/wt]) content of NaCl. Genes differentially expressed (p0.05) through the 48-h incubation were transcriptional regulators prfA and agrA, genes of the main virulence cluster inlA, hly, actA, involved in invasion of the epithelial cells, and genes bsh, opuC, gadC, clpP, and ami, associated with osmotic stress responses in L. monocytogenes. The more sensitive strain ATCC(®) 51779 was most responsive, showing significant upregulation of prfA, actA, hly, and bsh both at low and high NaCl. Strain DSMZ 15675 was less responsive to NaCl stress, showing reduced or consistent gene transcription at all conditions. Decreased transcription of agrA, ami, gadC, and opuC in both strains was promoted by low NaCl content. The study indicated that virulence gene expression of L. monocytogenes grown in cheese was affected by NaCl content and that effect was more significant in strains sensitive to both hypo- and hyperosmotic stresses.

Maari is a spontaneously fermented food condiment made from baobab tree seeds in West African countries. This type of product is considered to be safe, being consumed by millions of people on a daily basis. However, due to the spontaneous nature of the fermentation the human pathogen Bacillus cereus occasionally occurs in Maari. This study characterizes succession patterns and pathogenic potential of B. cereus isolated from the raw materials (ash, water from a drilled well (DW) and potash), seed mash throughout fermentation (0-96h), after steam cooking and sun drying (final product) from two production sites of Maari. Aerobic mesophilic bacterial (AMB) counts in raw materials were of 10(5)cfu/ml in DW, and ranged between 6.5×10(3) and 1.2×10(4)cfu/g in potash, 10(9)-10(10)cfu/g in seed mash during fermentation and 10(7) - 10(9) after sun drying. Fifty three out of total 290 AMB isolates were identified as B. cereus sensu lato by use of ITS-PCR and grouped into 3 groups using PCR fingerprinting based on Escherichia coli phage-M13 primer (M13-PCR). As determined by panC gene sequencing, the isolates of B. cereus belonged to PanC types III and IV with potential for high cytotoxicity. Phylogenetic analysis of concatenated sequences of glpF, gmk, ilvD, pta, pur, pycA and tpi revealed that the M13-PCR group 1 isolates were related to B. cereus biovar anthracis CI, while the M13-PCR group 2 isolates were identical to cereulide (emetic toxin) producing B. cereus strains. The M13-PCR group 1 isolates harboured poly-γ-D-glutamic acid capsule biosynthesis genes capA, capB and capC showing 99-100% identity with the environmental B. cereus isolate 03BB108. Presence of cesB of the cereulide synthetase gene cluster was confirmed by PCR in M13-PCR group 2 isolates. The B. cereus harbouring the cap genes were found in potash, DW, cooking water and at 8h fermentation. The "emetic" type B. cereus were present in DW, the seed mash at 48-72h of fermentation and in the final product, while the remaining isolates (PanC type IV) were detected in ash, at 48-72h fermentation and in the final product. This work sheds light on the succession and pathogenic potential of B. cereus species in traditional West African food condiment and clarifies their phylogenetic relatedness to B. cereus biovar anthracis. Future implementation of GMP and HACCP and development of starter cultures for controlled Maari fermentations will help to ensure a safe product.

Summary Background & aims This study is a part of the clinical trials with probiotic bacterium Lactobacillus salivarius Ls-33 conducted in obese adolescents. Previously reported clinical studies showed no effect of Ls-33 consumption on the metabolic syndrome in the subject group. The aim of the study was to investigate the impact of L . salivarius Ls-33 on fecal microbiota in obese adolescents. Methods The study was a double-blinded intervention with 50 subjects randomized to intake of L . salivarius Ls-33 or placebo for 12 weeks. The fecal microbiota was assessed by real-time quantitative PCR before and after intervention. Concentrations of fecal short chain fatty acids were determined using gas chromatography. Results Ratios of Bacteroides–Prevotella–Porphyromonas group to Firmicutes belonging bacteria, including Clostridium cluster XIV, Blautia coccoides _ Eubacteria rectale group and Roseburia intestinalis , were significantly increased ( p ≤ 0.05) after administration of Ls-33. The cell numbers of fecal bacteria, including the groups above as well as Clostridium cluster I, Clostridium cluster IV, Faecalibacterium prausnitzii , Enterobacteriaceae , Enterococcus , the Lactobacillus group and Bifidobacterium were not significantly altered by intervention. Similarly, short chain fatty acids remained unaffected. Conclusion L. salivarius Ls-33 might modify the fecal microbiota in obese adolescents in a way not related to metabolic syndrome. Clinical trial number: NCT 01020617

The connections between gut microbiota, energy homeostasis, and inflammation and its role in the pathogenesis of obesity-related disorders are increasingly recognized. We aimed to investigate the effect of the probiotic strain Lactobacillus salivarius Ls-33 on a series of biomarkers related to inflammation and the metabolic syndrome (MS) in adolescents with obesity.The study was a double-blind placebo-controlled trial including 50 adolescents with obesity randomized to Ls-33 (10 CFU) or placebo daily for 12 weeks.The average body mass index-for-age z-score was 2.6 ± 0.5. There were no differences in biomarkers of inflammation and parameters related to the MS at baseline between the probiotic and placebo groups. Furthermore, there were no differences in changes from baseline to 12-week intervention with regard to any anthropometric measures, blood pressure (systolic and diastolic), fasting glucose and insulin, homeostasis model assessment of insulin resistance, C-peptide, cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglyceride, free fatty acids, C-reactive protein, interleukin-6, tumor necrosis factor alpha, or fecal calprotectin, despite the increased values of biomarkers of inflammation and of several parameters related to the MS at baseline when compared with normal-weight adolescents. The levels of L salivarius in fecal samples from the probiotic group in the present study were comparable with the levels reported for the other probiotic lactobacilli and bifidobacteria using quantitative polymerase chain reaction.It was not possible to detect any beneficial effect of the probiotic intervention with Ls-33 on inflammatory markers or parameters related to the MS in adolescents with obesity being in a state of low-grade systemic inflammation.

According to animal studies, intake of probiotic bacteria may improve glucose homeostasis. We hypothesised that probiotic bacteria improve insulin sensitivity by attenuating systemic inflammation. Therefore, the effects of oral supplementation with the probiotic bacteriumLactobacillus acidophilusNCFM on insulin sensitivity and the inflammatory response were investigated in subjects with normal or impaired insulin sensitivity. In a double-blinded, randomised fashion, forty-five males with type 2 diabetes, impaired or normal glucose tolerance were enrolled and allocated to a 4-week treatment course with eitherL. acidophilusNCFM or placebo.L. acidophiluswas detected in stool samples by denaturating gradient gel electrophoresis and real-time PCR. Separated by the 4-week intervention period, two hyperinsulinaemic–euglycaemic clamps were performed to estimate insulin sensitivity. Furthermore, the systemic inflammatory response was evaluated by subjecting the participants toEscherichia colilipopolysaccharide injection (0·3 ng/kg) before and after the treatment course.L. acidophilusNCFM was detected in 75 % of the faecal samples after treatment with the probiotic bacterium. Insulin sensitivity was preserved among volunteers in theL. acidophilusNCFM group, whereas it decreased in the placebo group. Both baseline inflammatory markers and the systemic inflammatory response were, however, unaffected by the intervention. In conclusion, intake ofL. acidophilusNCFM for 4 weeks preserved insulin sensitivity compared with placebo, but did not affect the systemic inflammatory response.

BackgroundRecent evidence suggests that there is a link between metabolic diseases and bacterial populations in the gut. The aim of this study was to assess the differences between the composition of the intestinal microbiota in humans with type 2 diabetes and non-diabetic persons as control.Methods and FindingsThe study included 36 male adults with a broad range of age and body-mass indices (BMIs), among which 18 subjects were diagnosed with diabetes type 2. The fecal bacterial composition was investigated by real-time quantitative PCR (qPCR) and in a subgroup of subjects (N = 20) by tag-encoded amplicon pyrosequencing of the V4 region of the 16S rRNA gene. The proportions of phylum Firmicutes and class Clostridia were significantly reduced in the diabetic group compared to the control group (P = 0.03). Furthermore, the ratios of Bacteroidetes to Firmicutes as well as the ratios of Bacteroides-Prevotella group to C. coccoides-E. rectale group correlated positively and significantly with plasma glucose concentration (P = 0.04) but not with BMIs. Similarly, class Betaproteobacteria was highly enriched in diabetic compared to non-diabetic persons (P = 0.02) and positively correlated with plasma glucose (P = 0.04).ConclusionsThe results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota. The level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota.