L. pentosus L33 was originally isolated from fermented sausages (Pavli et al., 2016 (link)), and was acquired by the Institute of Technology of Agricultural Products, Hellenic Agricultural Organization DIMITRA (Athens, Greece). It was maintained in de Man, Rogosa, and Sharpe (MRS) broth (Condalab, Madrid, Spain) at 37°C for 16–18 h under anaerobic conditions, prior to DNA extraction. Bacterial cells were collected by centrifugation at 8,000 g for 4 min. Total genomic DNA was extracted from the cell pellets using the NucleoSpin® Tissue kit (Macherey-Nagel, Düren, Germany), according to manufacturer’s instructions. DNA purity and quantity were confirmed spectrophotometrically at 260 nm using NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States).
Mrs broth
Manufactured by Condalab
Sourced in Spain, United States
MRS broth is a culture medium used for the isolation and enumeration of lactic acid bacteria. It provides the necessary nutrients and growth factors required for the cultivation of Lactobacillus and other lactic acid-producing microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Nucleospin tissue kit, by Macherey-Nagel (4 mentions)
Mrs agar, by Condalab (3 mentions)
Ptrkh3 slpgfp vector, by Addgene (2 mentions)
In fusion method, by Takara Bio (2 mentions)
Erythromycin, by Merck Group (2 mentions)
Centrofriger bl 2, by J.P. Selecta (2 mentions)
Sterile quarter strength ringer s solution, by Thermo Fisher Scientific (2 mentions)
Yeast mold broth, by Condalab (2 mentions)
Glycerol, by Merck Group (1 mentions)
Luria bertani broth, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Glycerol, by AppliChem (1 mentions)
Nutrient broth, by Lab M (1 mentions)
L cysteine, by Merck Group (1 mentions)
Metformin, by Merck Group (1 mentions)
Mixed linkage β glucan kit, by Megazyme (1 mentions)
Glucose, by ITW Reagents (1 mentions)
Yeast extract, by MDBio (1 mentions)
Ferric ammonium sulfate, by Merck Group (1 mentions)
20 protocols using mrs broth
1
Isolation and DNA Extraction of Lactobacillus pentosus
2
Lc. lactis Recombinant Strain Characterization
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The bacteria used in this study were Lc. lactis AV1n isolated from a Tunisian avocado and the recombinant Lc. lactis AV1n[pRCR21] strain (Besrour-Aouam et al., 2019 (link)). The latter harbors the pRCR21 plasmid, which carries the PdsrLL–mrfp transcriptional fusion encoding the mCherry protein under the control of the promoter of the dsrLL gene of Lc. lactis AV1n. Also, pRCR21 carries the cat gene encoding the chloramphenicol acetyl transferase, which confers CmR to the bacterium. The bacteria were grown in MRS broth (Condalab, Torrejon de Ardoz, Madrid, Spain) supplemented with 2% glucose (MRSG) or with 2% sucrose (MRSS). In addition, the MRSS was supplemented with chloramphenicol (Cm) at 10 μg/ml, when Lc. lactis AV1n[pRCR21] was grown.
3
Engineered Probiotic NAPE-PLD Expression
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The pTRKH3-slpGFP vector (Addgene, Watertown, Massachusetts, United States) was first modified to remove the GFP sequence at SalI/PstI restriction sites, insert the T7 transcriptional terminator at BamHI/EcoRV sites, and insert the linker sequence containing BsaI-BsaI at PstI/XmaI restriction sites. The cDNA of human NAPE-PLD was then inserted into the BsaI sites using In-Fusion method (Clontech, Mountain View, CA, United States). The resulting pTRKH3-slp-NAPE-PLD and parental plasmid (not expressing NAPE-PLD gene, used as negative control) constructs were transfected into the L. paracasei subsp. paracasei F19 strain (Arla Foods, Hoersholm, Denmark) by electroporation, and positive clones were obtained by erythromycin (5 μg/ml) selection. Both parental plasmid (pLP) and NAPE-PLD expressing bacteria (pNAPE-LP) were amplified anaerobically in Man, Rogosa, and Sharpe (MRS) broth (Conda, Torrejón de Ardoz Madrid, Spain) and isolated in MRS agar (Conda, Torrejón de Ardoz Madrid, Spain) both supplemented with erythromycin 5 μg/ml (Sigma-Aldrich, Milan, Italy) under anaerobic conditions for 72 h at 37°C. Bacteria viability was determined by manually counting colonies, and the colony forming units (CFU)/ml was obtained through a colony number correction for the dilution factor.
4
Constructing NAPE-PLD Expressing Lactobacillus
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The pTRKH3-slpGFP vector (Addgene, Watertown, MA, USA) was first modified to remove the GFP sequence at SalI/PstI restriction sites, insert T7 transcriptional terminators at BamHI/EcoRV sites, and insert linker sequences containing BsaI-BsaI at PstI/XmaI restriction sites. The cDNA of human NAPE-PLD was then inserted into the BsaI sites using the In-Fusion method (Clontech, Mountain View, CA, USA). The resulting pTRKH3-slp-NAPE-PLD and parental plasmid (not expressing NAPE-PLD gene, used as negative control) constructs were transfected into the Lactobacillus paracasei subsp. paracasei F19 strain (Arla Foods, Hoersholm, Denmark) by electroporation, and positive clones were obtained by erythromycin (5 μg/mL) selection. Both parental plasmid (pLP) and NAPE-PLD-expressing bacteria (pNAPE-LP) were amplified anaerobically in Man, Rogosa and Sharpe (MRS)-broth (Conda, Torrejón de Ardoz Madrid, Spain) and isolated in MRS agar (Conda, Torrejón de Ardoz Madrid, Spain), both supplemented with erythromycin 5 μg/mL (Sigma-Aldrich, Milan, Italy) under anaerobic conditions for 72 h at 37 °C. Bacteria viability was determined by manually counting colonies, and the colony forming units (CFU)/mL were obtained through a colonies number correction for the dilution factor.
5
Enterocin-Mediated Inhibition of Listeria
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Enterococcus strains with enterocin genes were grown overnight in MRS broth (Condalab) at 37 °C under aerobic conditions. Two microliters of inoculums was spot inoculated onto the MRS agar plates and grown for 18 h at 37 °C under aerobic conditions. The MRS agar plates containing the growth of Enterococcus strains in spot form were then overlaid with 0.75% brain–heart infusion agar (Condalab) inoculated with 106 log cfu/mL of the different L. monocytogenes strains, and incubated at 37 °C for 24 h. Two independent trials were performed and each pathogenic strain was assayed in duplicate. The diameter of inhibition were measured and expressed in mm as the mean of n = 4.
6
DNA Extraction from Lactobacillus Strains
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Lc. paracasei SP5 was previously isolated from kefir grains (Mantzourani et al., 2019b (link)). Lc. rhamnosus GG ATCC 53103 (LGG) was acquired from DSMZ (Braunschweig, Germany). The bacterial strains were grown O/N in de Man, Rogosa and Sharp (MRS) broth (Condalab, Madrid, Spain) at 37°C, under anaerobic conditions. For DNA isolation, overnight cultures of Lc. paracasei SP5 were pelleted by centrifugation at 8,000 × g for 4 min. The pellet was lysed, and DNA was extracted using the NucleoSpin® Tissue kit (Macherey-Nagel, Düren, Germany), according to manufacturer’s instructions. DNA fragmentation was determined in 1% (w/v) agarose gel, and the quantity and quality of the isolated nucleic acids were also determined spectrophotometrically at 260 nm using NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States).
7
Isolation and Characterization of Algerian Fruit Lactobacilli
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Lactobacillus strains used in this work were recently isolated from Algerian fruits [31 (link)]. They include Lb. plantarum M10 and M12 isolated from blackberries (Rubus sp.), Lb. plantarum F2, F3 and 2F8 isolated from fresh figs (Ficus carica); Lb. plantarum NCA3, NCA4, FB3, FB13 and Lb. paracasei FB1 isolated from prickly pears (Opuntia ficus-indica). Stocks of these strains were maintained at −20 °C in de Man Rogosa and Sharpe (MRS) broth (Conda, Madrid, Spain), containing 30% (v/v) of glycerol (Sigma-Aldrich, Schnelldorf, Germany). These strains were cultivated anaerobically (AnaeroGen™ 2.5 L, Anaerobic Gas Generator, Oxoid, Thermo, Hampshire, UK) for 18–24 h in MRS broth at 37 °C prior use.
The target strains were E. coli ATCC 8739 isolated from feces, L. monocytogenes 162 isolated from food [32 (link)], and the clinical isolate S. aureus 2S6, kindly provided by Khalil Amrane hospital (Bejaia, Algeria). These strains were aerobically grown at 37 °C in brain heart infusion (BHI) (Sigma-Aldrich) or in Luria–Bertani (LB) broth (Sigma-Aldrich) and stored at −80 °C.
The target strains were E. coli ATCC 8739 isolated from feces, L. monocytogenes 162 isolated from food [32 (link)], and the clinical isolate S. aureus 2S6, kindly provided by Khalil Amrane hospital (Bejaia, Algeria). These strains were aerobically grown at 37 °C in brain heart infusion (BHI) (Sigma-Aldrich) or in Luria–Bertani (LB) broth (Sigma-Aldrich) and stored at −80 °C.
8
Isolation and Characterization of Lactobacillus Strains
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Lc. paracasei SRX10 was previously isolated from a traditional Greek cheese [7 (link)]. Lc. paracasei SP3 and Lc. paracasei SP5 were isolated from kefir grains [19 (link),20 (link)], and Lp. plantarum L125 was isolated from fermented sausages [21 (link)]. Strains Lc. rhamnosus GG and Lc. casei ATCC 393 were purchased from ATCC (LGC Standards, Middlesex, UK) and Lc. paracasei DSM 20,006 was obtained from DSMZ (Braunschweig, Germany). All strains were maintained in de Man, Rogosa, and Sharpe (MRS) broth (Condalab, Madrid, Spain) at 37 °C in anaerobic conditions. For DNA extraction, bacterial cells were harvested via centrifugation (8000× g for 4 min). The cell pellet was lysed, and DNA was extracted using the NucleoSpin® Tissue kit, following manufacturer’s instructions (Macherey-Nagel, Düren, Germany). DNA purity and quantity were determined spectrophotometrically (NanoDrop® ND-1000 UV–Vis Spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA).
9
Evaluation of Cava Lees as Bacterial Growth Promoter
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A scheme of the overall experimental procedure is provided in Figure 1 . In order to select the most effective concentration, the growth promoter effect of different amounts of cava lees was assessed in 6 bacterial strains of Latilactobacillus sakei (CTC494 and BAP111), Lactiplantibacillus plantarum subsp. plantarum (CECT4180 and CECT220), Latilactobacillus curvatus (BAP202), and Lacticaseibacillus casei (BAP341). An overnight culture of each strain was performed in MRS broth (Condalab®, Madrid, Spain) incubated in optimal growth conditions (Table 1 ). Then, a 100 µL aliquot from the overnight culture was subcultured in 25 mL of MRS broth without (control) or with increasing amounts of lees (0.5%, 1%, 2%, and 5% (w/v)) at the optimal temperature, and with constant shaking (200 rpm) under aerobic conditions. Amounts above 5% of lees were not considered, as they technically hindered the cultivation of the bacterial strains. Plate microbial counts, expressed as log10 CFU/mL, were determined in duplicate at incubation times 0 to 72 h. Three independent assays were performed for each bacterial strain.
10
Cultivation of L. plantarum MNC 21
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L. plantarum MNC 21 isolated from Obushera by Mukisa [23 ] was used. From the stock culture, 0.1 mL was delivered into 100 mL of sterile MRS broth (CONDA, Madrid, Spain) and incubated at 30˚C for 24 h. L. plantarum MNC 21 was subcultured thrice after which the cells were recovered by centrifugation at 7,500 x g for 10 min. The cell pellets were suspended in 10 mL of sterile Ringer's solution. Culture purity was checked using a microscope (020–518.500 DM/LS I/98 model, Leica, Germany).
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L. plantarum MNC 21 isolated from Obushera by Mukisa [23 ] was used. From the stock culture, 0.1 mL was delivered into 100 mL of sterile MRS broth (CONDA, Madrid, Spain) and incubated at 30˚C for 24 h. L. plantarum MNC 21 was subcultured thrice after which the cells were recovered by centrifugation at 7,500 x g for 10 min. The cell pellets were suspended in 10 mL of sterile Ringer's solution. Culture purity was checked using a microscope (020–518.500 DM/LS I/98 model, Leica, Germany).
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