Isolation, identification and characterisation of three novel probiotic strains (Lactobacillus paracaseiCNCM I-4034,Bifidobacterium breveCNCM I-4035 andLactobacillus rhamnosusCNCM I-4036) from the faeces of exclusively breast-fed infants

The aim of the present study was to isolate, identify and characterise novel strains of lactic acid bacteria and bifidobacteria with probiotic properties from the faeces of exclusively breast-fed infants. Of the 4680 isolated colonies, 758 exhibited resistance to low pH and tolerance to high concentrations of bile salts; of these, only forty-two exhibited a strong ability to adhere to enterocytesin vitro.The identities of the isolates were confirmed by 16S ribosomal RNA (rRNA) sequencing, which permitted the grouping of the forty-two bacteria into three different strains that showed more than 99 % sequence identity withLactobacillus paracasei,Lactobacillus rhamnosusandBifidobacterium breve, respectively. The strain identification was confirmed by sequencing the 16S–23S rRNA intergenic spacer regions. Strains were assayed for enzymatic activity and carbohydrate utilisation, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur and namedL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036. The strains were susceptible to antibiotics and did not produce undesirable metabolites, and their safety was assessed by acute ingestion in immunocompetent and immunosuppressed BALB/c mouse models. The three novel strains inhibitedin vitrothe meningitis aetiological agentListeria monocytogenesand human rotavirus infections.B. breveCNCM I-4035 led to a higher IgA concentration in faeces and plasma of mice. Overall, these results suggest thatL. paracaseiCNCM I-4034,B. breveCNCM I-4035 andL. rhamnosusCNCM I-4036 should be considered as probiotic strains, and their human health benefits should be further evaluated.

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In-vitro pathogen inhibition: Comparing the inhibitory effects of a complex multistrain synbiotic with simple probiotics containing the yeast Saccharomyces boulardii or Lactobacillus rhamnosus bacteria

10.1101/543819 ◽

2019 ◽

Author(s):

Jacek Piatek ◽

Henning Sommermeyer ◽

Arleta Ciechelska-Rybarczyk ◽

Malgorzata Bernatek

Keyword(s):

Bacterial Infections ◽

Pathogenic Bacteria ◽

Lactobacillus Rhamnosus ◽

Saccharomyces Boulardii ◽

Human Pathogens ◽

Inhibitory Effects ◽

Probiotic Strains ◽

Pathogen Inhibition ◽

Different Strains

AbstractSupplementation with probiotics is considered as alternative treatment or adjuvant therapy for a number of bacterial infections for which the use of antibiotics is either not recommended or emerging antibiotic resistance is a major concern. Inhibition of the growth of pathogenic bacteria has been related to a number of different activities of probiotic bacteria or yeasts, some of which are very specific for particular strains of probiotics. As the different inhibition activities might act additively or even synergistically, probiotic multistrain products are discussed as potentially being more effective in pathogen inhibition than products containing one or a small number of probiotic strains. The present study investigated the in vitro inhibition of Escherichia (E.) coli, Shigella spp., Salmonella (S.) typhimurium and Clostridum (Cl.) difficile, all being human pathogens of significant worldwide healthcare concerns. The probiotic containing the yeast Sacharomyces (S.) boulardii inhibited all four pathogens. Similar inhibitions were observed with a bacterial probiotic containing three different strains (Pen, E/N and Oxy) of Lactobacillus (Lc.) rhamnosus. Compared to the inhibition found for these probiotics, the inhibitory effects of a complex multistrain synbiotic, containing nine different probiotic strains (6 Lactobacilli and 3 Bifidobacteria) and the prebiotic fructooligosaccharide (FOS), were significantly stronger. The stronger inhibition by the complex multistrain synbiotic was observed for all four tested pathogens. Our findings support a hypothesis that complex synbiotic products containing a larger number of different strains combined with a prebiotic component might be more attractive candidates for further clinical characterization than simpler probiotics containing one or only few probiotic strains.

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Lactobacillus rhamnosus GG Genomic and Phenotypic Stability in an Industrial Production Process

Applied and Environmental Microbiology ◽

10.1128/aem.02780-19 ◽

2020 ◽

Vol 86 (6) ◽

Author(s):

Marianne Stage ◽

Anita Wichmann ◽

Mette Jørgensen ◽

Natalia Ivonne Vera-Jimenéz ◽

Malue Wielje ◽

...

Keyword(s):

Production Process ◽

Gene Cluster ◽

Industrial Production ◽

Lactobacillus Rhamnosus ◽

Probiotic Properties ◽

Phenotypic Stability ◽

Content Type ◽

Freeze Dried ◽

Probiotic Strains

ABSTRACT Lactobacillus rhamnosus GG is one of the most widely marketed and studied probiotic strains. In L. rhamnosus GG, the spaCBA-srtC1 gene cluster encodes pili, which are important for some of the probiotic properties of the strain. A previous study showed that the DNA sequence of the spaCBA-srtC1 gene cluster was not present in some L. rhamnosus GG variants isolated from liquid dairy products. To examine the stability of the L. rhamnosus GG genome in an industrial production process, we sequenced the genome of samples of L. rhamnosus GG (DSM 33156) collected at specific steps of the industrial production process, including the culture collection stock, intermediate fermentations, and final freeze-dried products. We found that the L. rhamnosus GG genome sequence was unchanged throughout the production process. Consequently, the spaCBA-srtC1 gene locus was intact and fully conserved in all 31 samples examined. In addition, different production batches of L. rhamnosus GG exhibited consistent phenotypes, including the presence of pili in final freeze-dried products, and consistent characteristics in in vitro assays of probiotic properties. Our data show that L. rhamnosus GG is highly stable in this industrial production process. IMPORTANCE Lactobacillus rhamnosus GG is one of the best-studied probiotic strains. One of the well-characterized features of the strain is the pili encoded by the spaCBA-srtC1 gene cluster. These pili are involved in persistence in the gastrointestinal tract and are important for the probiotic properties of L. rhamnosus GG. Previous studies demonstrated that the L. rhamnosus GG genome can be unstable under certain conditions and can lose the spaCBA-srtC1 gene cluster. Since in vitro studies have shown that the loss of the spaCBA-srtC1 gene cluster decreases certain L. rhamnosus GG probiotic properties, we assessed both the genomic stability and phenotypic properties of L. rhamnosus GG throughout an industrial production process. We found that neither genomic nor phenotypic changes occurred in the samples. Therefore, we demonstrate that L. rhamnosus GG retains the spaCBA-srtC1 cluster and exhibits excellent genomic and phenotypic stability in the specific industrial process examined here.

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ISOLATION, CLASSIFICATION AND IDENTIFICATION OF POTENTIAL PROBIOTIC LACTOBACILLUS STRAINS FROM INFANT FAECES

Science and Technology Development Journal ◽

10.32508/stdj.v14i4.2049 ◽

2011 ◽

Vol 14 (4) ◽

pp. 62-76

Author(s):

Khanh Quoc Hoang ◽

Thanh Thi Lan Pham

Keyword(s):

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Probiotic Properties ◽

Specific Pcr ◽

Probiotic Lactobacillus ◽

Probiotic Strains ◽

Pcr Method ◽

Breast Fed ◽

Rdna Analysis

Lactobacillus bacteria present in many probiotic products. This paper investigated probiotic Lactobacillus strains isolated from the human gastrointestinal tract. 15 Lactobacillus strains were isolated from breast-fed infant faeces and identified by both traditional methods and genus-specific PCR method. In vitro experiments were designed to investigate some probiotic properties such as resistance to low pH and bile, cell surface hydrophobicity, antimicrobial activity, bacteriocin and other antimicrobials production, antibiotic resistance and cholesterol reduction. As a result, 12 probiotic Lactobacillus strains were selected. Significantly, 11 strains of them reduced 10-33.34 % serum cholesterol level. By 16S rDNA analysis, the probiotic strains were classified at species level as Lactobacillus gasseri, L. fermentum, L. salivarius, L. rhamnosus and L. paracasei/ casei.

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Probiotic properties of native Lactobacillus spp. strains for dairy calves

Beneficial Microbes ◽

10.3920/bm2017.0131 ◽

2018 ◽

Vol 9 (4) ◽

pp. 613-624 ◽

Cited By ~ 4

Author(s):

S. Fernández ◽

M. Fraga ◽

E. Silveyra ◽

A.N. Trombert ◽

A. Rabaza ◽

...

Keyword(s):

Lactobacillus Reuteri ◽

Bacterial Species ◽

Control Group ◽

Probiotic Properties ◽

Real Time Quantitative Pcr ◽

Treatment And Prevention ◽

Probiotic Strains ◽

Lactobacillus Spp

The use of native microorganisms with probiotic capacity is an alternative tool for the treatment and prevention of several diseases that affect animals, such as neonatal calf diarrhoea. The selection of probiotic strains within a collection is based on different in vitro and in vivo assays, which predict their potential. The aim of this study was to characterise a group of native Lactobacillus spp. strains isolated from faeces of healthy calves using an in vitro approach and to assess their ability to colonise the gastrointestinal tract (GIT) of calves. Native Lactobacillus spp. strains were evaluated on their capacity to survive low pH conditions and bile salts presence, biofilm formation and adhesion to both mucus and Caco-2 cells. Based on the in vitro characterisation, four strains (Lactobacillus johnsonii TP1.1, Lactobacillus reuteri TP1.3B, L. johnsonii TP1.6 and Lactobacillus amylovorus TP8.7) were selected to evaluate their capacity to colonise and persist in the GIT of calves. The assessment of enteric persistence involved an in vivo assay with oral administration of probiotics and quantification in faeces of the administered bacterial species with real-time quantitative PCR (qPCR). The study was conducted using 15 calves (1-month-old) which were divided into five groups of three animals, four of which were treated with four different selected strains and one was the control group. Strains TP1.3B and TP1.6 managed to persist in treated animals until ten days after the end of the administration period, indicating that they could be promising candidates for the design of probiotics for calves.

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Intraspecific Genotypic Characterization of Lactobacillus rhamnosus Strains Intended for Probiotic Use and Isolates of Human Origin

Applied and Environmental Microbiology ◽

10.1128/aem.00091-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5376-5383 ◽

Cited By ~ 29

Author(s):

M. Vancanneyt ◽

G. Huys ◽

K. Lefebvre ◽

V. Vankerckhoven ◽

H. Goossens ◽

...

Keyword(s):

Lactobacillus Rhamnosus ◽

Probiotic Strain ◽

Starter Cultures ◽

Probiotic Properties ◽

Human Origin ◽

Culture Collections ◽

Clinical Case Studies ◽

Probiotic Strains ◽

Different Sources

ABSTRACT A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.

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Selection and Evaluation of Potential Probiotic Lactic Acid Bacteria and Bifidobacteria Isolated from Human Intestinal Tract

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.910.137 ◽

2014 ◽

Vol 910 ◽

pp. 137-140

Author(s):

Chao Hui Xue ◽

Lan Wei Zhang ◽

Hong Bo Li ◽

Shu Mei Wang

Keyword(s):

Lactobacillus Rhamnosus ◽

Antimicrobial Activities ◽

E Coli ◽

Probiotic Potential ◽

Probiotic Lactic Acid Bacteria ◽

Probiotic Strains ◽

Health Promoting ◽

Culture Supernatants ◽

Ht 29

Three Lactobacillus strains were screened on the basis of probiotic characteristics (i.e., resistance to low pH and bile salts, adhesion to the human gastrointestinal tract, inhibition of pathogenic strains). They further exhibited producing antimicrobial activities of non-acid molecule (s). In addition, antibacterial peptides were isolated and purified from the cell-free culture supernatants of these three probiotic strains. Based on TricineSDSPAGE, the antimicrobial peptide was approximately 10 kDa in size. After analyzing the sequence of the 16SrDNA regions of these three strains, they were identified asLactobacillus crispatus Lactobacillus rhamnosus and Lactobacillus rhamnosua GG.Using an in vitro system simulating gastric transit, our findings indicated that the three probiotic strains had the ability to tolerate gastroenteric environment and the adhesive capacity to HT-29 cells. It was demonstrated that the probiotic strains inhibited subsequent adhesion of E. coli to the HT-29 cell. Among the selected strains,L. rhamnosusF1333 showed a high probiotic potential and could be used in health-promoting food products.

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Lactobacillus rhamnosus attenuates Thai chili extracts induced gut inflammation and dysbiosis despite capsaicin bactericidal effect against the probiotics, a possible toxicity of high dose capsaicin

PLoS ONE ◽

10.1371/journal.pone.0261189 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0261189

Author(s):

Wimonrat Panpetch ◽

Peerapat Visitchanakun ◽

Wilasinee Saisorn ◽

Ajcharaporn Sawatpanich ◽

Piraya Chatthanathon ◽

...

Keyword(s):

Gene Expression ◽

Tight Junction ◽

Healthy Volunteers ◽

Lactobacillus Rhamnosus ◽

High Dose ◽

Fluorescent Staining ◽

Qrt Pcr ◽

Junction Molecules ◽

High Concentrations

Because of a possible impact of capsaicin in the high concentrations on enterocyte injury (cytotoxicity) and bactericidal activity on probiotics, Lactobacillus rhamnosus L34 (L34) and Lactobacillus rhamnosus GG (LGG), the probiotics derived from Thai and Caucasian population, respectively, were tested in the chili-extract administered C57BL/6 mice and in vitro experiments. In comparison with placebo, 2 weeks administration of the extract from Thai chili in mice caused loose feces and induced intestinal permeability defect as indicated by FITC-dextran assay and the reduction in tight junction molecules (occludin and zona occludens-1) using fluorescent staining and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the chili extracts also induced the translocation of gut pathogen molecules; lipopolysaccharide (LPS) and (1→3)-β-d-glucan (BG) and fecal dysbiosis (microbiome analysis), including reduced Firmicutes, increased Bacteroides, and enhanced total Gram-negative bacteria in feces. Both L34 and LGG attenuated gut barrier defect (FITC-dextran, the fluorescent staining and gene expression of tight junction molecules) but not improved fecal consistency. Additionally, high concentrations of capsaicin (0.02–2 mM) damage enterocytes (Caco-2 and HT-29) as indicated by cell viability test, supernatant cytokine (IL-8), transepithelial electrical resistance (TEER) and transepithelial FITC-dextran (4.4 kDa) but were attenuated by Lactobacillus condition media (LCM) from both probiotic-strains. The 24 h incubation with 2 mM capsaicin (but not the lower concentrations) reduced the abundance of LGG (but not L34) implying a higher capsaicin tolerance of L34. However, Lactobacillus rhamnosus fecal abundance, using qRT-PCR, of L34 or LGG after 3, 7, and 20 days of the administration in the Thai healthy volunteers demonstrated the similarity between both strains. In conclusion, high dose chili extracts impaired gut permeability and induced gut dysbiosis but were attenuated by probiotics. Despite a better capsaicin tolerance of L34 compared with LGG in vitro, L34 abundance in feces was not different to LGG in the healthy volunteers. More studies on probiotics with a higher intake of chili in human are interesting.

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Probiotic Lactobacillus Strains Stimulate the Inflammatory Response and Activate Human Macrophages

Journal of Immunology Research ◽

10.1155/2017/4607491 ◽

2017 ◽

Vol 2017 ◽

pp. 1-14 ◽

Cited By ~ 37

Author(s):

L. M. Rocha-Ramírez ◽

R. A. Pérez-Solano ◽

S. L. Castañón-Alonso ◽

S. S. Moreno Guerrero ◽

A. Ramírez Pacheco ◽

...

Keyword(s):

Innate Immunity ◽

Bactericidal Activity ◽

Nuclear Translocation ◽

Lactobacillus Rhamnosus ◽

Human Macrophages ◽

E Coli ◽

Probiotic Lactobacillus ◽

Tlr2 Activation ◽

Probiotic Strains ◽

Different Strains

Lactobacilli have been shown to promote health functions. In this study, we analyzed the mechanism by which four different strains of probiotics affected innate immunity, such as regulation of ROS, cytokines, phagocytosis, bactericidal activity, signaling by NF-κB pp65, and TLR2 activation. The production of ROS was dependent on the concentration and species of Lactobacillus. The results obtained from the tested strains (Lactobacillus rhamnosus GG, L. rhamnosus KLSD, L. helveticus IMAU70129, and L. casei IMAU60214) showed that strains induced early proinflammatory cytokines such as IL-8,TNF-α, IL-12p70, and IL-6. However, IL-1β expression was induced only by L. helveticus and L. casei strains (after 24 h stimulation). Phagocytosis and bactericidal activity of macrophages against various pathogens, such as S. aureus, S. typhimurium, and E. coli, were increased by pretreatment with Lactobacillus. The nuclear translocation NF-κB pp65 and TLR2-dependent signaling were also increased by treatment with the probiotics. Taken together, the experiments demonstrate that probiotic strains of Lactobacillus exert early immunostimulatory effects that may be directly linked to the initial inflammation of the response of human macrophages.

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Identification of Bifidobacterium Animalis Ssp. Lactis from Egyptian Women Breast Milk and Feces of Breast Fed Infant Based On 16S-23S rRNA Gene

Advances in Nutrition & Food Science ◽

10.33140/anfs/01/01/00009 ◽

2016 ◽

Vol 1 (1) ◽

Keyword(s):

Dna Sequences ◽

Patent Protection ◽

Health Benefit ◽

Intergenic Spacer ◽

23S Rrna ◽

Species Discrimination ◽

Rrna Gene ◽

Accession Number ◽

Intergenic Spacer Region ◽

Breast Fed

Bifidobacterium represent one of the major genera of the intestinal tract of human and animals used as probiotics in dairy and nondairy foods for restore the intestinal microflora which confers a health benefit. The identification of Bifidobacterium by phenotypic features is commonly unreliable, time, money, and effort consuming. We sought to improve the Bifidobacterium identification method based on molecular level to identify probiotic bacteria in complex microbial communities. The application of 16S-23S rRNA oligonucleotide primers is the best and most reliable, rapid, and precise species and sub species identification approach. The ribosomal intergenic spacer region (ISR) located between the highly conserved 16S rRNA and 23S rRNA shows a high degree of variation in length and sequence and potential for intra species discrimination and providing the phylogenetic Relationship of the Genus Bifidobacterium spp. Results showed that one of the two primer sets Bflac2-Bflac5 species specific gives positive results differentiating between B. animalis ssp. Lactis isolated from breast fed infants milk of human and that isolated from feces of breast fed infant and detecting reference strain for B. animalis ssp. Lactis DSM10140. DNA sequences of the two strains were submitted to the Genbank NCBI under accession number (KT758845) named as B. animalis ssp. Lactis Egm1 (Egyptian milk) and accession number (KT758846) named as Egf1 Egyptian feces while the second primer give false positive result. Also, we aim to obtain patent protection under Intellectual property rights (IPRs) for B. animalis ssp. Lactis which was isolated from Egyptian resources to be used for a better and healthier food and dairy products.

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Characterization of Lactobacillus species proposed as probiotics

Potravinarstvo Slovak Journal of Food Sciences ◽

10.5219/1479 ◽

2021 ◽

Vol 15 ◽

pp. 143-150

Author(s):

Saad Sabah Fakhry ◽

Farqad Abdullah Rashid ◽

Maha Muhamaed Khudiar ◽

Lubna Ayad Ismail ◽

Sarah Khattab Ismail ◽

...

Keyword(s):

Antimicrobial Activity ◽

Lactobacillus Rhamnosus ◽

Lactobacillus Species ◽

Probiotic Properties ◽

Biochemical Tests ◽

Survival Percentage ◽

Sds Page ◽

Vitro Analysis

An isolated Lactobacillus from several various sources were identified depending on morphological, microscopically and biochemical tests in vitro analysis of probiotic properties that included: an ability to tolerate in different concentration of bile salt, survival in acidic conditions, their antimicrobial activity, and S-layer characterizations were carried out. It was noticed that isolates of Lactobacillus rhamnosus and L. delbrueckii have a broad activity of antimicrobial and found the isolate L. rhamnosus represented with a survival percentage 6.9% at pH 4.5 and 5.1% at pH 2.0) also L. rhamnosus (5.7% at pH 4.5 and 4.9% at pH 2.0) tolerated acidic media, Lactobacillus spp. has antimicrobial activity against all gram-positive and negative tested isolates. 70 kDa of S-layer protein bands were detected with whole-cell SDS-PAGE analysis, and it's predominant in cells of isolates which grown in MRS broth anaerobically. It was noticed that the collected Lactobacillus isolates could be used as probiotic.

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