Isolation and identification of a human intestinal bacterium capable of daidzein conversion

2021 ◽  
Author(s):  
Yingyu Guo ◽  
Lichao Zhao ◽  
Xiang Fang ◽  
Qingping Zhong ◽  
Huijun Liang ◽  
...  
Keyword(s):  
Bacterial Population ◽  
Quantitative Polymerase Chain Reaction ◽  
Complex Mixture ◽  
Antioxidant Response ◽  
Growth Media ◽  
Intestinal Bacterium ◽  
Propidium Monoazide ◽  
Isolation And Identification ◽  
Healthy Human ◽  
Polymerase Chain

Abstract Equol, which produced from daidzein (one of the principal isoflavones), is recognized to be the most resultful in stimulating an estrogenic and antioxidant response. The daidzein transformation was studied during fermentation of five growth media inoculated with feces from a healthy human, and a daidzein conversion strain was isolated. To enrich the bacterial population involved in daidzein metabolism in a complex mixture, fecal samples were treated with antibiotics. The improved propidium monoazide combined with the quantitative polymerase chain reaction (PMAxx-qPCR) assay showed that the ampicillin treatment of samples did result in a reduction of the total visible bacteria counts by 52.2% compared to the treatment without antibiotics. On this basis, the newly isolated rod-shaped, gram-positive anaerobic bacterium, named strain Y11 (MN560033), was able to metabolize daidzein to equol under anaerobic conditions, with a conversion ratio (equol ratio: the amount of equol produced/amount of supplemented daizein) of 0.56 over 120 h. The 16S rRNA partial sequence of the strain Y11 exhibited 99.8% identity to that of Slackia equolifaciens strain DZE (NR116295). This study will provide new insights into the biotransformation of equol from daidzein by intestinal microbiota from the strain-level and explore the possibility of probiotic interventions.

scholarly journals Identification of aflatoxigenic fungi using polymerase chain reaction-based assay

2014 ◽  
pp. 259-269 ◽  
Author(s):  
Vladislava Soso ◽  
Marija Skrinjar ◽  
Nevena Blagojev ◽  
Slavica Veskovic-Moracanin
Keyword(s):  
Polymerase Chain Reaction ◽  
Regulatory Gene ◽  
Control Point ◽  
Complex Mixture ◽  
Target Sequence ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Close Link ◽  
Critical Control Point ◽  
Polymerase Chain

As the aflatoxins represent a health-risk for humans because of their proven carcinogenicity, food-borne fungi that produce them as secondary metabolites, mainly Aspergillus flavus and Aspergillus parasiticus, have to be isolated and identified. The best argument for identifying problem fungi is that it indicates control points within the food system as part of a hazard analysis critical control point (HACCP) approach. This assumes there is a close link between fungus and toxin. Conventional methods for isolation and identification of fungi are time consuming and require admirably dedicated taxonomists. Hence, it is imperative to develop methodologies that are relatively rapid, highly specific and as an alternative to the existing methods. The polymerase chain reaction (PCR) facilitates the in vitro amplification of the target sequence. The main advantages of PCR is that organisms need not be cultured, at least not for a long time, prior to their detection, target DNA can be detected even in a complex mixture, no radioactive probes are required, it is rapid, sensitive and highly versatile. The gene afl-2 has been isolated and shown to regulate aflatoxin biosynthesis in A. flavus. Also, the PCR reaction was targeted against aflatoxin synthesis regulatory gene (aflR1) since these genes are nearly identical in A. flavus and A. parasiticus in order to indicate the possibility of detection of both the species with the same PCR system (primers/reaction). [Projekat Ministarstva nauke Republike Srbije, br. III46009] <br><br><font color="red"><b> This article has been retracted. Link to the retraction <u><a href="http://dx.doi.org/10.2298/APT1647265E">10.2298/APT1647265E</a><u></b></font>

scholarly journals The effect of starter culture on bacterial profile in soybean tempeh

Food Research ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 380-389
Author(s):  
T.E. Pramudito ◽  
E.G.A. Putri ◽  
E. Paluphi ◽  
Y. Yogiara
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacterial Population ◽  
Quantitative Polymerase Chain Reaction ◽  
Starter Culture ◽  
Chain Reaction ◽  
Medium Scale ◽  
Three Samples ◽  
Polymerase Chain ◽  
Processing Techniques ◽  
Total Bacteria

The bacterial population in soybean tempeh can be affected by many factors such as hygiene and preparation procedure of the soybean. In this study, we investigate the effect of different tempeh starter culture on the bacterial profile in comparison to other factors. The bacterial profile of five commercial tempeh made with different processing techniques and three samples made with different starter culture were analyzed using quantitative polymerase chain reaction (qPCR). The phylum Verrucomicrobia was only detected in commercial tempeh from unstandardized home-scale industries. Bacteroidetes were present in commercial tempeh and samples made with starter that contains cassavabased materials. Commercial tempeh from standardized medium-scale industries contains fewer total bacteria than those from home-scale industries. The addition of starter culture for tempeh production can increase the number of total bacteria compared to uninoculated soybeans. However, there was no difference in total bacterial amount between starter culture variation. Bacterial profiles of all samples were dominated by Firmicutes except tempeh with the starter culture Cap Jago that is dominated by Gammaproteobacteria. This study showed that although the type of starter culture did not significantly contribute to total bacteria in tempeh, it can influence the bacterial profile of the product on phylum level.

scholarly journals Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova

2017 ◽  
Vol 75 (11) ◽  
pp. 2615-2621 ◽  
Author(s):  
P. Gyawali ◽  
J. P. S. Sidhu ◽  
W. Ahmed ◽  
P. Jagals ◽  
S. Toze
Keyword(s):  
Environmental Samples ◽  
Quantitative Measurement ◽  
Quantitative Polymerase Chain Reaction ◽  
Detection Methods ◽  
Quantitative Detection ◽  
Propidium Monoazide ◽  
Chain Reaction ◽  
Vital Stain ◽  
Polymerase Chain ◽  
Hookworm Ova

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P &lt; 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

scholarly journals Propidium monoazide combined with qPCR to differentiate live and dead conidia of Neofabraea actinidiae

2018 ◽  
Vol 71 ◽  
pp. 348
Author(s):  
Lucia Ramos ◽  
I.P. Shamini Pushparajah ◽  
M. Shahjahan Kabir ◽  
Bethan E. Parry ◽  
Kerry R. Everett
Keyword(s):  
Light Emitting Diode ◽  
Quantitative Polymerase Chain Reaction ◽  
Propidium Monoazide ◽  
Chain Reaction ◽  
Light Emitting ◽  
Qpcr Analysis ◽  
Low Concentrations ◽  
Polymerase Chain ◽  
High Concentrations ◽  
Commercial Kit

Neofabraea actinidiae can occasionally cause post-harvest rot in kiwifruit. Quantitative polymerase chain reaction (qPCR) analysis represents a feasible and accurate option for identifying and quantifying this rot but is limited because qPCR results do not differentiate live and dead conidia. Propidium monoazide (PMA) is a photoreactive dye that penetrates into the damaged cell-wall membranes of dead conidia binding to the DNA and thus suppressing its amplification by qPCR. A commercial kit containing PMA was trialled for differentiating between live and dead N. actinidiae conidia. The most suitable conditions were 1 μM PMA with 10 min light emitting diode (LED) exposure, and could clearly distinguish high concentrations of live from similar concentrations of dead conidia when tested separately and as a mixture. Low concentrations of live N. actinidiae conidia could be distinguished from dead ones when tested separately, but not as a mixture. Additional work is needed to optimise the effectiveness of the PMA binding and apply this concept in the orchard.

scholarly journals Rapid and Accurate Detection of Bacteriophage Activity againstEscherichia coliO157:H7 by Propidium Monoazide Real-Time PCR

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Hui Liu ◽  
Yan D. Niu ◽  
Jinquan Li ◽  
Kim Stanford ◽  
Tim A. McAllister
Keyword(s):  
Cell Surface ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Extracellular Dna ◽  
Bacterial Cells ◽  
Propidium Monoazide ◽  
Direct Plating ◽  
E Coli ◽  
Polymerase Chain ◽  
Phage Activity

Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol ofE. colirequire several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intactE. colicells that survive phage exposure.Escherichia coliO157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cellsE. coliO157:H7. Compared to PMA-qPCR, direct plating overestimated (P< 0.01) phage efficacy as cell surface-attached phage particles lysedE. coliO157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol ofE. coliO157:H7.

scholarly journals Propidium Monoazide Improves Quantification of Resting Spores of Plasmodiophora brassicae with qPCR

Plant Disease ◽  
2017 ◽  
Vol 101 (3) ◽  
pp. 442-447 ◽  
Author(s):  
Fadi Al-Daoud ◽  
Bruce D. Gossen ◽  
Justin Robson ◽  
Mary Ruth McDonald
Keyword(s):  
Heat Treatment ◽  
Plasmodiophora Brassicae ◽  
Quantitative Polymerase Chain Reaction ◽  
Infested Soil ◽  
Propidium Monoazide ◽  
Spore Viability ◽  
Heat Treated ◽  
Resting Spores ◽  
Polymerase Chain ◽  
The Impact

Plasmodiophora brassicae, which causes clubroot of Brassica crops, persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) analysis is often used to quantify resting spores but does not distinguish between DNA of viable and nonviable spores. The impact of pretreating spores with propidium monoazide (PMA), which inhibits amplification of DNA from nonviable microorganisms, was assessed in several experiments. Spore suspensions from immature and mature clubs were heat treated; then, PMA-PCR analyses and bioassays were performed to assess spore viability. Prior to heat treatment, assessments comparing PMA-PCR to qPCR for mature spores were similar, indicating that most of these spores were viable. However, only a small proportion (<26%) of immature spores were amplified in PMA-PCR. Bioassays demonstrated that clubroot severity was much higher in plants inoculated with mature spores than with immature spores. Heat treatment produced little or no change in estimates of mature spores from qPCR but spore estimates from PMA-PCR and clubroot severity in bioassays were both substantially reduced. Estimates of spore concentration with PMA-PCR were less consistent for immature spores. To facilitate use of PMA-PCR on infested soil, a protocol for extracting spores from soil was developed that provided higher extraction efficiency than the standard methods.

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