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

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.

scholarly journals 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

2013 ◽  
Vol 109 (S2) ◽  
pp. S51-S62 ◽  
Author(s):  
Sergio Muñoz-Quezada ◽  
Empar Chenoll ◽  
José María Vieites ◽  
Salvador Genovés ◽  
José Maldonado ◽  
...  
Keyword(s):  
Lactobacillus Rhamnosus ◽  
Intergenic Spacer ◽  
23S Rrna ◽  
Strain Identification ◽  
Probiotic Properties ◽  
Probiotic Strains ◽  
High Concentrations ◽  
Different Strains ◽  
Breast Fed

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.

scholarly journals Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase and d-Cycloserine Synthetase Found in the Biosynthetic Pathway

2013 ◽  
Vol 57 (6) ◽  
pp. 2603-2612 ◽  
Author(s):  
Narutoshi Uda ◽  
Yasuyuki Matoba ◽  
Takanori Kumagai ◽  
Kosuke Oda ◽  
Masafumi Noda ◽  
...  
Keyword(s):  
Gene Cluster ◽  
High Performance ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Homocysteine Thiolactone ◽  
Putative Gene ◽  
Content Type ◽  
Dna Fragment ◽  
Layer Chromatography

ABSTRACTWe have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic,d-cycloserine. The gene cluster is composed of 10 open reading frames, designateddcsAtodcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization ofO-ureidoserine. DcsD is similar toO-acetylserine sulfhydrylase, which generatesl-cysteine usingO-acetyl-l-serine with sulfide, and therefore, DcsD may be a synthase to generateO-ureido-l-serine usingO-acetyl-l-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase convertingO-ureido-d-serine intod-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed inEscherichia coliand purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substratesO-acetyl-l-serine and hydroxyurea, synthesis ofd-cycloserine was successfully attained. Thesein vitrostudies yield the conclusion that DcsD and DcsG are necessary for the syntheses ofO-ureido-l-serine andd-cycloserine, respectively. DcsD was also able to catalyze the synthesis ofl-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclicd-amino acid analogs, such asd-homocysteine thiolactone.

scholarly journals Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics

2013 ◽  
Vol 79 (6) ◽  
pp. 1923-1933 ◽  
Author(s):  
François P. Douillard ◽  
Angela Ribbera ◽  
Hanna M. Järvinen ◽  
Ravi Kant ◽  
Taija E. Pietilä ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Transcription Initiation ◽  
Lactobacillus Rhamnosus ◽  
Comparative Genomic ◽  
Phenotypic Analysis ◽  
Carbohydrate Utilization ◽  
Content Type ◽  
Host Signaling ◽  
Toll Like Receptor 2 ◽  
Genome Content

ABSTRACTFourLactobacillusstrains were isolated from marketed probiotic products, includingL. rhamnosusstrains from Vifit (Friesland Campina) and Idoform (Ferrosan) andL. caseistrains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail withL. caseistrain BL23 andL. rhamnosusstrain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization betweenL. caseiandL. rhamnosusstrains, which could be linked to their genotypes. The two isolatedL. rhamnosusstrains had genomes that were virtually identical to that ofL. rhamnosusGG, testifying to their genomic stability and integrity in food products. TheL. caseistrains showed much greater genomic heterogeneity. Remarkably, all strains contained an intactspaCBApilus gene cluster. However, only theL. rhamnosusstrains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of aniso-IS30element upstream of the pilus gene cluster inL. rhamnosusstrains but absent inL. caseistrains had constituted a functional promoter driving pilus gene expression. AllL. rhamnosusstrains triggered an NF-κB response via Toll-like receptor 2 (TLR2) in a reporter cell line, whereas theL. caseistrains did not or did so to a much lesser extent. This study demonstrates that the twoL. rhamnosusstrains isolated from probiotic products are virtually identical toL. rhamnosusGG and further highlights the differences between these andL. caseistrains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities.

scholarly journals The Gene Cluster forpara-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98

2014 ◽  
Vol 80 (19) ◽  
pp. 6212-6222 ◽  
Author(s):  
Jun Min ◽  
Jun-Jie Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Gene Knockout ◽  
Gene Clusters ◽  
Sole Source ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Biochemical Analyses ◽  
Nitrophenol Degradation

ABSTRACTBurkholderiasp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) orpara-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of thepnpgenes in thepnpABA1CDEFcluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activityin vitroin the conversion of 2C4NP to CBQ. Genetic analyses indicated thatpnpAplays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.

Probiotic properties of native Lactobacillus spp. strains for dairy calves

2018 ◽  
Vol 9 (4) ◽  
pp. 613-624 ◽  
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.

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

2006 ◽  
Vol 72 (8) ◽  
pp. 5376-5383 ◽  
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.

scholarly journals Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy

2015 ◽  
Vol 81 (17) ◽  
pp. 5794-5803 ◽  
Author(s):  
Komlavi Anani Afanou ◽  
Anne Straumfors ◽  
Asbjørn Skogstad ◽  
Ajay P. Nayak ◽  
Ida Skaar ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Field Emission ◽  
Polyclonal Antibodies ◽  
Detection Methods ◽  
Indoor Environments ◽  
Content Type ◽  
Freeze Dried ◽  
Scanning Electron

ABSTRACTSubmicronic fungal fragments have been observed inin vitroaerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores fromAspergillus versicolorand high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed withA. versicolorfragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments ofA. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.

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

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.

scholarly journals 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

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.

scholarly journals Topical Probiotics for Women’s Urogenital Health: Selection of an Oil-based Carrier

2021 ◽  
Vol 06 (04) ◽  
pp. 1-1
Author(s):  
Scarlett Puebla-Barragan ◽  
◽  
Britney Lamb ◽  
Serenah Jafelice ◽  
Gregor Reid ◽  
...  
Keyword(s):  
Lactobacillus Reuteri ◽  
Lactobacillus Rhamnosus ◽  
Coconut Oil ◽  
Petroleum Jelly ◽  
Vaginal Microbiome ◽  
Topical Product ◽  
Freeze Dried ◽  
Probiotic Strains ◽  
Selection Of

Vaginal care products are widely used by women to relieve discomfort such as pain, itching and malodour, all of which are commonly caused by conditions resulting from microbiota dysbiosis. Previous studies showed that probiotic strains Lacticaseibacillus (formerly Lactobacillus) rhamnosus GR-1 (LGR-1) and Limosilactobacillus (formerly Lactobacillus) reuteri RC-14 (LRC-14), can aid in restoring homeostasis in the vaginal microbiome when taken orally. A topical product containing these strains could be of value for reducing malodour and improving quality of life. However, the formulation of such a product is a challenge, given that its ingredients must maintain shelf-life viability by excluding moisture. Here, we tested petroleum jelly, mineral oil, coconut oil, and olive oil for how well they maintained the viability of freeze-dried probiotic strains over a six-month timeframe. None of the oils caused excessive loss of bacterial viability, with petroleum jelly and coconut oil showing the most promise. Based on existing knowledge of these oils on the female genitalia, coconut oil and petroleum jelly could be suitable probiotic carriers for clinical testing.

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