Purpose
Ability to survive the digestive process is a major factor in determining the effectiveness of a probiotic. In this study, the ability of the probiotic L. casei DG
®
(
Lactobacillus paracasei
...CNCMI1572) to survive gastrointestinal transit in healthy children was investigated for the first time.
Methods
Twenty children aged 3–12 years received L. casei DG
®
as drinkable solution of 1 × 10
9
colony forming units (CFU), once daily for 7 consecutive days. Recovery in faecal samples was evaluated at baseline and at different time-points during and after administration. Defecation frequency, faeces consistency, digestive function and product safety were also assessed.
Results
Nineteen (95%) of the 20 enrolled children presented viable L. casei DG
®
cells in their faeces at least once during the study, with a maximum count (mean 4.3 log
10
CFU/g ± 2.3) reached between day 4 and 6 from the beginning of consumption. Notably, for 11 (57.9%) of the 19 children with viable cells, L. casei DG
®
survived in faecal samples up to 3 days after treatment end. Defecation frequency, faeces consistency and digestive function did not change considerably during or after study treatment. Safety of the study product was very good.
Conclusions
This study showed for the first time that L. casei DG
®
survives the gastrointestinal transit when ingested by children with a paediatric probiotic drinkable solution containing 1 × 10
9
CFU, and persists in the gut up to 3 days after the end of product intake, demonstrating resistance to gastric juices, hydrolytic enzymes and bile acids.
This study aimed to predict the optimal carbon source for higher production of exopolysaccharides (EPS) by Lactobacillus paracasei TD 062, and to evaluate the effect of this carbon source on the ...production and monosaccharide composition of EPS. We evaluated the EPS production capacity of 20 strains of L. paracasei under the same conditions. We further investigated L. paracasei TD 062, which showed the highest EPS-producing activity (0.609 g/L), by examining the associated biosynthesis pathways for EPS. Genomics revealed that fructose, mannose, trehalose, glucose, galactose, and lactose were carbon sources that L. paracasei TD 062 could use to produce EPS. We identified an EPS synthesis gene cluster that could participate in transport, export, and sugar chain synthesis, and generate 6 sugar nucleotides. Experimental results showed that the sugar content of the EPS produced using fermentation with the optimized carbon source (fructose, mannose, trehalose, glucose, galactose, and lactose) increased by 115%. Furthermore, use of the optimized carbon source changed the monosaccharide content of the associated EPS. The results of enzyme activity measurements showed significant increases in the activity of 2 key enzymes involved in the glycoside synthesis pathway. Our study revealed that optimizing the carbon source provided for fermentation not only increased the production of EPS, but also affected the composition of the monosaccharides by increasing enzyme activity in the underlying synthesis pathways, suggesting an important role for carbon source in the production of EPS by L. paracasei TD 062.
The mechanisms of bacterial adhesion to human cells involve several complex reactions and activation of genes and proteins. It has been reported that the food components in dairy matrices, such as ...sugar or salt, can decrease bacterial adhesion to Caco-2 cells. However, it has not been evaluated whether the bacteria grown in media supplemented with milk phospholipids (MPL) can increase or decrease the adhesion of these cells. The objective of this work was to evaluate the effects of MPL on the kinetic growth of lactic acid bacteria (LAB) and their functional characteristics as probiotics, expression of surface protein genes, and adherence to Caco-2 cells. Seven LAB strains isolated from various dairy products were characterized. Five of the tested LAB strains were able to grow in a chemically defined medium supplemented with MPL. Lactobacillus reuteri OSU-PECh-48 showed the highest growth rate and the greatest optical density. All of the strains tested showed tolerance to acidic conditions at pH 3.0 and to bile salts at 0.5 and 1% concentrations. Auto-aggregation and cell surface hydrophobicity ability were evaluated, with nonsignificant differences between the strains grown in MPL and without MPL. Gene expression of 6 surface proteins was evaluated in the presence or absence of MPL. Pediococcus acidilactici OSU-PECh-L and OSU-PECh-48 were the strains with highest relative expression of 5 of the 6 genes evaluated. Lactobacillus paracasei OSU-PECh-BA was the strain with the lowest level of expression of surface protein genes. Most of the bacteria tested had increased adhesion to Caco-2 cells after growth in MPL. The bacteria with the highest degrees of adhesion observed were Lactobacillus paracasei OSU-PECh-3B, Pediococcus acidilactici OSU-PECh-L, and Lactobacillus reuteri OSU-PECh-48. The genes Cnb and EF-Tu increased in expression in the presence of MPL in most of the LAB tested. The results obtained in this work demonstrate the high potential of these LAB strains for use as starters or beneficial cultures in fermentation of not only dairy products but also other food fermentation processes, with promising ability to increase residence time in the gut, modify the microbiome, and improve human health.
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•VL8-EPS promoted the cell proliferation and upregulated the mRNA expression of proinflammatory cytokines such as IL-1β, TNF-α, IL-6 and IL-10.•RAW264.7 macrophages mainly recognized ...polysaccharides through TLR4.•VL8-EPS promoted nuclear translocation of the p65 subunit of NF-κB.•VL8-EPS stimulated RAW264.7 macrophages through the upregulation of the NF-κB and MAPK signaling pathways.
This study explored the immunoregulatory mechanism of VL8-EPS, obtained from Viili. An RAW264.7 macrophage cell-line was used along with real-time quantitative PCR to evaluate the impact of VL8-EPS on mRNA expression of IL-1β, TNF-α, IL-6, IL-10, and TLR4. Moreover, western blot assay was conducted to study the signal pathway of NF-κB and MAPK. The VL8-EPS was found to upregulate the mRNA expression of these cytokines and TLR4, while it promoted protein phosphorylation of p65, IκBα, ERK1/2, and JNK. The immunofluorescence staining and inhibitor blocking experiments proved that TLR4, NF-κB, and MAPK signal pathway were involved to secrete immune-enhancing substances. In conclusion, VL8-EPS was involved to activate downstream signal pathways of NF-κB and MAPK through TLR4 identification, which caused an increase in mRNA expression and cytokines release, ultimately exerting immune regulation.
Serpa cheese is one of the traditional regional Portuguese cheeses having the Protected Denomination of Origin (PDO) designation. This study investigated the bacterial community in the traditional ...Portuguese Serpa cheese. The microorganisms identified at the end of ripening (30 days) mainly were lactic acid bacteria (LAB). Lactobacillus paracasei/Lactobacillus casei was the main species in cheese from PDO registered industries, whereas in non‐PDO registered industries Lactobacillus brevis was highlighted, among other LAB. Enterobacteriaceae species were detected at 20% to 40% of the total isolates. The results obtained by high‐throughput sequencing analysis confirmed that LAB was the main microbial group, with Lactococcus genus contributing to approximately 40% to 60% of the population, followed by Leuconostoc and Lactobacillus. The Enterobacteriaceae family was also important. The differences between bacterial communities from PDO and non‐PDO registered industries suggest that the lack of regulation of the cheese‐making practices may influence unfavorably. The new knowledge about bacterial diversity in Serpa cheese could be useful to set up new ripening conditions, which favor the development of desirable microorganisms.
Practical Application
The control of the manufacturing process of traditional cheeses can be improved through the knowledge of the bacterial diversity that develops. Thus, the growth of desirable microorganisms can be promoted to homogenize the final product.
Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD) whose exact cause is still unclear. Disruption of the intestinal microflora is considered one of the main causes of the disease. ...Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) is a multifunctional strain that has been shown in previous studies to possess anti-inflammatory properties and to exert a modulatory effect on intestinal bacteria associated with certain pathogenic mechanisms of IBD. In the current study, we investigated the effects of NTU 101 on dextran sulfate sodium (DSS)-induced colitis in a mouse model. Colitis was induced in male C57BL/6 mice (total number n = 60) via dissolved DSS in drinking water on days 15–21 of the experiment. The effects of continuous 25 d feeding (days 0–25) of either a half or a full dose 2.3 × 109 colony-forming units (CFU)/kg body weight (BW)/d and 4.5 × 109 CFU/kg BW/d, respectively of NTU 101 was evaluated. Lactobacillus rhamnosus BCRC 16000 (BCRC 16000) and L. paracasei subsp. paracasei BCRC 14023 (BCRC 14023) strains were given to control groups. The results indicated that NTU 101 powder improved anti-oxidant capacity, reduced pro-inflammatory cytokine levels, increased anti-inflammatory cytokine levels, and slightly ameliorated body weight loss in DSS-treated mice during the final days of the study. This indicated that NTU 101 powder can relieve the clinical symptoms of DSS-induced colitis in mice.
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•In this study, in vivo model of colitis to explore the effect of NTU 101 on the clinical symptoms of ulcerative colitis.•NTU 101 administration alleviated the weight loss by DSS and increased the amount of intestinal Bifidobacterium spp.•It reduced the oxidative stress caused by DSS and hindered the secretion of proinflammatory cytokines in the colon.•NTU 101 may be potentially developed into an adjuvant therapy to prevent the recurrence of colitis in patients.
Palmitoylethanolamide (PEA) is an
-acylethanolamide produced on-demand by the enzyme
-acylphosphatidylethanolamine-preferring phospholipase D (NAPE-PLD). Being a key member of the larger family of ...bioactive autacoid local injury antagonist amides (ALIAmides), PEA significantly improves the clinical and histopathological stigmata in models of ulcerative colitis (UC). Despite its safety profile, high PEA doses are required in vivo to exert its therapeutic activity; therefore, PEA has been tested only in animals or human biopsy samples, to date. To overcome these limitations, we developed an NAPE-PLD-expressing
(pNAPE-LP), able to produce PEA under the boost of ultra-low palmitate supply, and investigated its therapeutic potential in a murine model of UC. The coadministration of pNAPE-LP and palmitate led to a time-dependent release of PEA, resulting in a significant amelioration of the clinical and histological damage score, with a significantly reduced neutrophil infiltration, lower expression and release of pro-inflammatory cytokines and oxidative stress markers, and a markedly improved epithelial barrier integrity. We concluded that pNAPE-LP with ultra-low palmitate supply stands as a new method to increase the in situ intestinal delivery of PEA and as a new therapeutic able of controlling intestinal inflammation in inflammatory bowel disease.
Lactobacillus paracasei is able to persist in a variety of natural and technological environments despite physico-chemical perturbations, in particular alternations between desiccation and ...rehydration. However, the way in which it adapts to hydric fluctuations and the genetic determinants involved are not clearly understood. To identify the genes involved in adaptation to desiccation, an annotated library of L. paracasei random transposon mutants was screened for viability after desiccation (25% relative humidity, 25 °C). We found 16 genes that have not been described as being involved in this response. Most of them are linked to either the transport of molecules or to cell wall structure and function. Our screening also identified genes encoding DNA related enzymes and an alarmone necessary for L. paracasei survival. Subsequently, the expression of the identified genes was measured at five stages of the dehydration-rehydration process to decipher the chronology of genetic mechanisms. They were classified into four different transcriptional profiles: genes upregulated during both desiccation and rehydration phases, genes upregulated during the desiccation phase only, genes downregulated during both desiccation and rehydration and genes downregulated only during the rehydration stage. Thus, genetic response to hydric fluctuations seems to occur during desiccation and can continue or not during rehydration. The genes identified should contribute to improve the stabilization of Lactobacillus starters in dry state.
•16 as yet unidentified genes are necessary for L. paracasei survival to desiccation.•Half of the identified genes are up regulated in the wild type during hydric stress.•Chronology of transcription of the 16 genes showed four transcriptomic profiles.•Dehydration is a critical phase for L. paracasei response to hydric fluctuations.
Lactic acid bacteria are broadly employed as starter cultures in the manufacture of foods. Upon technological preparation, they are confronted with drying stress that amalgamates numerous stress ...conditions resulting in losses of fitness and survival. To better understand and differentiate physiological stress responses, discover general and specific markers for the investigated stress conditions, and predict optimal preconditioning for starter cultures, we performed a comprehensive genomic and quantitative proteomic analysis of a commonly used model system, Lactobacillus paracasei subsp. paracasei TMW 1.1434 (isogenic with F19) under 11 typical stress conditions, including among others oxidative, osmotic, pH, and pressure stress. We identified and quantified >1900 proteins in triplicate analyses, representing 65% of all genes encoded in the genome. The identified genes were thoroughly annotated in terms of subcellular localization prediction and biological functions, suggesting unbiased and comprehensive proteome coverage. In total, 427 proteins were significantly differentially expressed in at least one condition. Most notably, our analysis suggests that optimal preconditioning toward drying was predicted to be alkaline and high-pressure stress preconditioning. Taken together, we believe the presented strategy may serve as a prototypic example for the analysis and utility of employing quantitative-mass-spectrometry-based proteomics to study bacterial physiology.
The disturbance of intestinal microorganisms and the exacerbation of type 2 diabetes (T2D) are mutually influenced. In this study, the effect of exopolysaccharides (EPS) from
JY039 on the adhesion of
...JY062 was investigated, as well as their preventive efficacy against T2D. The results showed that the EPS isolated from
JY039 effectively improved the adhesion rate of
JY062 to Caco-2 cells (1.8 times) and promoted the proliferation of
JY062. In the mice experiment, EPS,
JY062 and their complex altered the structure of the intestinal microbiota, which elevated the proportion of
,
, while inversely decreasing the proportion of
,
and other bacteria involved in energy metabolism (
< 0.01;
< 0.05); enhanced the intestinal barrier function; promoted secretion of the gut hormone peptide YY (PYY) and glucagon-like peptide-1 (GLP-1); and reduced inflammation by balancing pro-inflammatory factors IL-6, TNF-α and anti-inflammatory factor IL-10 (
< 0.01;
< 0.05). These results illustrate that EPS and
JY062 have the synbiotic potential to prevent and alleviate T2D.
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