The lactic acid bacteria strain was isolated from the gastrointestinal tracts of compassionately sacrificed weaned piglet of the indigenous South African Windsnyer pig breed (APIEC13/008), and were cultured in de Man–Rogosa–Sharpe broth (Oxoid, Hampshire, UK), under strict anaerobic conditions. A pure culture of the isolate in de Man–Rogosa–Sharpe broth was washed in phosphate buffer saline (pH 7) and the bacterial genomic DNA was extracted from the washed cells with a DNA extraction kit (Zymo Research, Irvine, CA, USA). The purity and concentration of the extracted genomic DNA were determined using a nanodrop spectrophotometer (NanoDrop 2000, ThermoFisher, Germiston, South Africa). The identification of the isolate was confirmed through PCR amplification of the 16S rRNA region [13 (link)], and the resultant PCR amplicon was sequenced. The partial sequenced data (1552 bp) were, thereafter, submitted to the GenBank data base, and an accession number (MK123485) was received (https://www.ncbi.nlm.nih.gov/nuccore/MK123485.1/ ).
De man rogosa sharpe broth
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
De Man Rogosa Sharpe (MRS) Broth is a microbiological culture medium used for the selective isolation and enumeration of Lactobacillus species. It provides the necessary nutrients and growth factors to support the cultivation of these bacteria.
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Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Dna extraction kit, by Zymo Research (2 mentions)
Anaerogen system, by Thermo Fisher Scientific (1 mentions)
Protocatechuic, by Merck Group (1 mentions)
Hydroxycinnamic acid, by Merck Group (1 mentions)
Milli q purification system, by Merck Group (1 mentions)
1 2 dihydroxybenzene, by Merck Group (1 mentions)
3 phenyllactic acid, by Merck Group (1 mentions)
Magnesium sulphate mgso4, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Sinapic acid, by Merck Group (1 mentions)
Ferulic acid, by Merck Group (1 mentions)
Benzoic acid, by Merck Group (1 mentions)
Syringic acid, by Merck Group (1 mentions)
Vanillin, by Merck Group (1 mentions)
Lactic acid, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Potato dextrose agar (pda), by Liofilchem (1 mentions)
Buffered peptone water (bpw), by Liofilchem (1 mentions)
Potato dextrose broth (pdb), by Liofilchem (1 mentions)
9 protocols using de man rogosa sharpe broth
1
Isolation and Identification of Indigenous Lactic Acid Bacteria
2
Isolation and Characterization of Probiotic Bacterial Strain
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The candidate probiotic bacterial strain was isolated from the gastrointestinal tracts of compassionately sacrificed weaned piglets of the indigenous South African Windsnyer pig breed (APIEC13/008). The organism was cultured in de Man-Rogosa-Sharpe broth (Oxoid, UK), under strict anaerobic conditions and incubated at 37°C for 24 hours in an anaerobic jar provided with an AnaeroGen system (Thermofisher, UK). The culture was later washed twice in phosphate buffer saline (PBS) and centrifuge at 6000 rpm for 5 minutes each time. Bacterial genomic DNA was extracted with a DNA extraction kit (Zymo Research, USA), in accordance with the manufacturer’s instructions. The degree of purity and concentration of the extracted DNA were determined using a nanodrop spectrophotometer (NanoDrop 2000, ThermoFisher). The genomic DNA material was kept at −20°C for further use.
3
Quantitative Analysis of Phenolic Compounds
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The FB1 standard solution (purity > 99%) was obtained from Sigma–Aldrich (St. Louis, MO, USA). The phenolic standards 1,2-dihydroxybenzene, 3,4-dihydroxicinnamic acid, benzoic acid, 3-phenylLactic acid, hydroxycinnamic acid, p-coumaric acid, protocatechuic, sinapic acid, vanillin, syringic acid, and ferulic acid were purchased from Sigma–Aldrich (St. Louis, MO, USA). Lactic acid was obtained from Sigma–Aldrich (St. Louis, MO, USA).
Acetonitrile (ACN) (LC-MS/MS grade), ethyl acetate (EA), formic acid (FA), and methanol (HPLC-MS/MS grade) were obtained from VWR Chemicals (Randor, PA, USA). The deionised water used in chromatography analysis (<18 MΩ cm resistivity) was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA). The salts, magnesium sulphate (MgSO4) and sodium chloride (NaCl), were provided from Sigma–Aldrich (St. Louis, MO, USA).
The Potato Dextrose Agar (PDA), Potato Dextrose Broth (PDB), and Buffered peptone water (BPW) were purchased from Liofilchem Bacteriology Products (Roseto, Italy). De man Rogosa Sharpe (MRS) Broth was obtained from Oxoid (Hampshire, UK).
The Yellow Mustard Flour (YM) (code #106) and Oriental Mustard Flour (OM) (code #107) were provided by G.S. Dunn Dry Mustard Millers (Hamilton, ON, Canada).
Acetonitrile (ACN) (LC-MS/MS grade), ethyl acetate (EA), formic acid (FA), and methanol (HPLC-MS/MS grade) were obtained from VWR Chemicals (Randor, PA, USA). The deionised water used in chromatography analysis (<18 MΩ cm resistivity) was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA). The salts, magnesium sulphate (MgSO4) and sodium chloride (NaCl), were provided from Sigma–Aldrich (St. Louis, MO, USA).
The Potato Dextrose Agar (PDA), Potato Dextrose Broth (PDB), and Buffered peptone water (BPW) were purchased from Liofilchem Bacteriology Products (Roseto, Italy). De man Rogosa Sharpe (MRS) Broth was obtained from Oxoid (Hampshire, UK).
The Yellow Mustard Flour (YM) (code #106) and Oriental Mustard Flour (OM) (code #107) were provided by G.S. Dunn Dry Mustard Millers (Hamilton, ON, Canada).
4
Isolation and Characterization of Lactobacillus casei
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L. casei Imunitass DN-114001 strains were isolated from a fermented
milk product (ACTIMEL, DANONE, Spain)® and cultured in DeMan-Rogosa-Sharpe (MRS)
broth (Oxoid, England) for 24 h at 30 °C and a pH around 5.5.
The growing L. casei colonies were subjected to the following
identification: morphological identification, direct Gram strain, and biochemical
reactions including the glucose fermentation test and oxidase test, and catalase
production. It was stored in MRS broth at −80 °C till use. Isolated L.
casei were subcultured twice in MRS broth for 24 h at 30 °C prior to the
viable count. Lactobacilli were counted by serial dilutions and
subcultured onto MRS agar plates under the same incubation conditions. The pH for
each culture was measured after 0, 1, 2, 3, 4, 5, 6, and 24 h of incubation.
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L. casei Imunitass DN-114001 strains were isolated from a fermented
milk product (ACTIMEL, DANONE, Spain)® and cultured in DeMan-Rogosa-Sharpe (MRS)
broth (Oxoid, England) for 24 h at 30 °C and a pH around 5.5.
The growing L. casei colonies were subjected to the following
identification: morphological identification, direct Gram strain, and biochemical
reactions including the glucose fermentation test and oxidase test, and catalase
production. It was stored in MRS broth at −80 °C till use. Isolated L.
casei were subcultured twice in MRS broth for 24 h at 30 °C prior to the
viable count. Lactobacilli were counted by serial dilutions and
subcultured onto MRS agar plates under the same incubation conditions. The pH for
each culture was measured after 0, 1, 2, 3, 4, 5, 6, and 24 h of incubation.
5
Antimicrobial Activity Screening of Chicory Samples
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Antimicrobial activity was measured using the liquid culture method in a total volume of 1 mL (Puupponen-Pimiä et al. 2016 (link)). We screened the following microbial strains: Staphylococcus aureus VTT E-70045 (ATCC 6538), Pseudomonas aeruginosa VTT E-84219 (ATCC 15692), Lactobacillus rhamnosus GG VTT E-96666 (ATCC 53103) and Streptococcus thermophilus VTT E-96665 (ATCC 19258). S. aureus and P. aeruginosa were cultivated aerobically in BD Difco nutrient broth (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C, shaking at 150 rpm. L. rhamnosus and S. thermophilus were cultivated in De Man Rogosa Sharpe (MRS) broth (Oxoid, Basingstoke, UK) at 37 °C without agitation. Microbial stock cultures were stored at − 80 °C. Before cultivation, they were grown on solid media for 1–2 days as described above for each strain. Single colonies were transferred to liquid media, incubated for 20–24 h, and used as the source of inoculum for antimicrobial activity tests. Microbial cultures incubated with enzyme(s) in 100 µL acetic acid buffer without antimicrobial samples were used as positive growth controls. Cultures were incubated with 50 µg/mL chloramphenicol in 100 µL acetic acid buffer as a negative control. The inhibitory effects of chicory samples suspended in 100 µL acetic acid buffer were evaluated by comparison with the control growth curves.
6
Characterization of Food-Grade LAB Strains
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A total of 446 food-grade LAB strains (with a currently qualified presumption of safety—QPS—status as judged by the EFSA scientific Panels), mainly from lactobacilli, Pediococcus spp. and Leuconostoc spp., all belonging to the Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Bari, Italy, and the Faculty of Science and Technology, Free University of Bolzano, Bolzano, Italy, were investigated in this study. All strains were previously isolated from animals (Apis milifera intestine, Drosophila melanogaster intestine), dairy products (cheese, milk), cereals (spelt, oat, and tritordeum), fruits and vegetables (apple, avocado, carrot, cherry, fennel, grape, kiwi, olives, papaya, pineapple, prune, sauerkraut, and tomato), sourdough, and other sources (Table S3 ). Cultures were maintained as frozen stocks at −20 °C in De-Man-Rogosa-Sharpe (MRS) broth (Oxoid, Basingstoke, Hampshire, UK) medium with 20% glycerol for subsequent analysis. Before their use, the bacteria were propagated twice in MRS brothat 30 °C for 24 h.
7
Isolation and Characterization of Lactobacillus Strains
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The bacterial strains used in this study were L. delbrueckii TU-1, L. paracasei KTN-5, and
L. plantarum 22A-3 (Table 1 Bacterial strains used in this study
dietary fermented food products that were commercially available in Japan and that their taxonomic identities were confirmed by
our species-specific quantitative PCR assay, which will be described later. All bacterial isolates were stored in de
Man-Rogosa-Sharpe (MRS) broth (Oxoid, Basingstoke, United Kingdom) at −80°C until used.
L. plantarum 22A-3 (
| Strain | Origin |
| Lactobacillus delbrueckii TU-1 | Yogurt commercially available in Japan |
| Lactobacillus paracasei KTN-5 | Fermented milk product commercially available in Japan |
| Lactobacillus plantarum 22A-3 | Nukaduke (rice bran pickling) of eggplants |
dietary fermented food products that were commercially available in Japan and that their taxonomic identities were confirmed by
our species-specific quantitative PCR assay, which will be described later. All bacterial isolates were stored in de
Man-Rogosa-Sharpe (MRS) broth (Oxoid, Basingstoke, United Kingdom) at −80°C until used.
8
Isolation and Culture of L. crispatus
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L. crispatus
9
Glycomacropeptide Isolation and Purification
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Glycomacropeptide, with a maximum assayed lactose content of 1%, was provided by Agropur Ingredients, (Eden Prairie, MN). Vivaspin 6 centrifugal filters with a 3-kDa molecular weight cutoff were purchased from Sartorius Stedim Biotech GmbH (Göttingen, Germany). Bifidobacterium longum ssp. infantis ATCC 15697 was purchased from DSMZ (Braunschweig, Germany).
de Man, Rogosa, Sharpe (MRS) broth was purchased from Oxoid Ltd. (Basingstoke, UK). The Anaerocult A system was purchased from Merck (Darmstadt, Germany). All other reagents were from Sigma-Aldrich Co. (Dublin, Ireland), unless otherwise stated, and were of the highest grade available.
de Man, Rogosa, Sharpe (MRS) broth was purchased from Oxoid Ltd. (Basingstoke, UK). The Anaerocult A system was purchased from Merck (Darmstadt, Germany). All other reagents were from Sigma-Aldrich Co. (Dublin, Ireland), unless otherwise stated, and were of the highest grade available.
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