Maximum recovery diluent

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom, Ireland

Maximum recovery diluent is a specialized liquid medium designed for the reconstitution and dilution of samples in laboratory settings. It is formulated to maximize the recovery of target analytes, ensuring accurate and reliable results during analytical procedures.

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25 protocols using maximum recovery diluent

Microbial Analysis of Food Samples

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For microbiological analyses, 25 g of Control sample was diluted with 100 mL Maximum Recovery Diluent (Oxoid, Basingstoke, UK) and homogenised for 1 min in a Stomacher (PBI International, Milan, Italy). By contrast, Ground, Pre-homogenized, HPH 80, HPH 150 and HPH 150x10 samples (Table 1)
were directly used. Serial dilutions of each suspension were made in Maximum Recovery Diluent (Oxoid) and analysed for microbial counts. Appropriate aliquots (0.1 or 1 g) were spread on agar plates.
Plate Count Agar (Oxoid) and Man Ragosa Sharpe (MRS) were used for enumeration of total bacterial count and lactic acid bacteria respectively, and plates were incubated for 48 h at 30 °C. Oxytracycline-Glucose-Yeast Extract (OGY) agar (Oxoid), was used for enumeration of yeasts, and plates were incubated for 72 h at 28 °C.

Plazzotta S, & Manzocco L. (2019). High-pressure homogenisation combined with blanching to turn lettuce waste into a physically stable juice. Innovative Food Science and Emerging Technologies., 52.

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Probiotic Strain Survival in Gut

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Fecal samples from day 14, corresponding to the last day of the probiotic consumption (the first subsequent evacuation available was considered for a few samples when no evacuation occurred on the 14th day), were processed to verify the ability of the four probiotic strains to survive gastrointestinal transit. For this purpose, one gram of fecal sample was diluted in Maximum Recovery Diluent (Oxoid, Basingstoke, UK), homogenized in a sterile stomacher bag by using a Colworth Stomacher 400 instrument (Seward, West Sussex, UK) and plated on MRS supplemented with 0.05% cysteine and 5 µg mL⁻¹ streptomycin (scMRS). At least five dilutions per fecal sample were plated. After 48 h of incubation at 37 °C in anaerobic conditions with the use of Anaerocult A (Merck, Kenilworth, NJ, USA), all the colonies from each dilution plate were collected separately, and the biomass from the respective dilution plates was used for total DNA isolation as described above. Afterwards, qPCR with probiotic-specific primers was performed on the DNA isolated from the colony biomasses. The highest dilution giving a positive signal in qPCR and the obtained Cq value were used to calculate the “estimated minimum CFU number” (eCFU) for each investigated strain.

Taverniti V., Koirala R., Dalla Via A., Gargari G., Leonardis E., Arioli S, & Guglielmetti S. (2019). Effect of Cell Concentration on the Persistence in the Human Intestine of Four Probiotic Strains Administered through a Multispecies Formulation. Nutrients, 11(2), 285.

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Isolation and Characterization of Lactic Acid Bacteria

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Utilising a maximum recovery diluent (Oxoid, Basingstoke, UK), a ten-fold dilution series was performed on samples of fermented milk. Selected dilutions were applied in triplicates on MRS agar (Biolife, Milan, Italy) and then incubated in jars with Anaerogen (Oxoid, Basingstoke, UK) under anaerobic conditions for 48 h. After incubation, representative colonies from each plate were chosen and purified on MRS agar several times. The isolation was achieved on MRS agar by analysing morphological parameters (colony and cell morphology), as well as using biochemical assays (Gram staining and catalase test) [22 (link)]. Until further investigation, LAB characteristic colonies (Gram-positive, catalase-negative) were kept at 80 °C in MRS broth (Oxoid, Basingstoke, UK), which was supplemented with 30% glycerol.

Vasiliauskaite A., Mileriene J., Kasparaviciene B., Aleksandrovas E., Songisepp E., Rud I., Axelsson L., Muizniece-Brasava S., Ciprovica I., Paskevicius A., Aksomaitiene J., Gabinaitiene A., Uljanovas D., Baliukoniene V., Lutter L., Malakauskas M, & Serniene L. (2023). Screening for Antifungal Indigenous Lactobacilli Strains Isolated from Local Fermented Milk for Developing Bioprotective Fermentates and Coatings Based on Acid Whey Protein Concentrate for Fresh Cheese Quality Maintenance. Microorganisms, 11(3), 557.

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Quantification of Bifidobacterium and Lactobacillus in Faeces and Milk

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Aliquots of faecal sample, 1 gram, were mixed with 9 ml maximum recovery diluent (Oxoid) to make an initial 10⁻¹ dilution. Serial dilutions were spread-plated onto de Man, Rogosa, Sharpe (MRS) (Difco) agar supplemented with 0.05% L-cysteine hydrochloride (Sigma), 100 μg ml⁻¹ mupirocin (Oxoid) and 50 Units nystatin (Sigma Aldrich) for Bifidobacterium spp. and MRS agar (Difco) supplemented with 50 U ml⁻¹ nystatin for Lactobacillus spp. 1 ml of milk was serially diluted and spread-plated as described above. Agar plates were incubated anaerobically at 37 °C for 72 hours for Bifidobacterium spp. and 5 days for Lactobacillus spp. Bacterial counts were recorded as colony forming units per gram of faeces or per ml of human-milk.

Murphy K., Curley D., O’Callaghan T.F., O’Shea C.A., Dempsey E.M., O’Toole P.W., Ross R.P., Ryan C.A, & Stanton C. (2017). The Composition of Human Milk and Infant Faecal Microbiota Over the First Three Months of Life: A Pilot Study. Scientific Reports, 7, 40597.

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Isolation and Detection of EPEC and STEC

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All RAMS were transported on ice to the laboratory of UGent, stored overnight at 4 °C, and homogenized in 25 mL of maximum recovery diluent (Oxoid, Dilbeek, Belgium) by stomaching [20 (link)]. At ULiège, the whole procedure to isolate EPEC and (AE-)STEC was slightly adapted from Thiry and collaborators [17 (link)].
Briefly, 100 µL of the suspension was added to 5 mL of lauryl sulfate broth (Sigma-Aldrich, Darmstadt, Germany) and incubated overnight at 37 °C. Bacterial DNA was extracted from 1.5 mL of the enrichment broths via the alkaline-boiling method and stored at −20 °C. Lysates were tested with a triplex PCR targeting the eae, stx1, and stx2 genes [21 (link)]. Each PCR-positive broth was subsequently streaked onto four (semi-)selective agar plates and incubated overnight at 37 °C: McConkey’s (MC), Chromocult Coliform ES (ES) (VWR, Leuven, Belgium), Chromocult Coliform ES supplemented with 2.5 mg/mL of potassium tellurite (ESTe) (Sigma-Aldrich, Darmstadt, Germany) [22 (link)], and supplemented CHROMagarTM STEC base (STECB) (I2A, Montpellier, France).

Habets A., Engelen F., Duprez J.N., Devleesschauwer B., Heyndrickx M., De Zutter L., Thiry D., Cox E, & Mainil J. (2020). Identification of Shigatoxigenic and Enteropathogenic Escherichia coli Serotypes in Healthy Young Dairy Calves in Belgium by Recto-Anal Mucosal Swabbing. Veterinary Sciences, 7(4), 167.

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Antimicrobial Susceptibility of A. acidoterrestris

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First, A. acidoterrestris cells were grown overnight on Potato dextrose agar (Difco, BD) at 43 °C. The colonies were suspended in 10 mL Maximum Recovery Diluent (Oxoid) to obtain a bacterial density of McFarland 1.0 (10⁶ CFU/mL) using a densitometer (Den-1, HVD Life Sciences, Austria). After centrifugation at 16,000 × g for 5 min, the pellet was dissolved in 10 mL apple juice. Then, 180 µL juice containing antimicrobial (nisin 0–100 IU/mL or lysozyme, 0–100 mg/L) and 20 µL of the bacterial suspension were added into the wells of a flat bottom 96-well plate in duplicates (Corning Costar) for each temperature tested. The plates were incubated individually at 27, 35 or 43 °C in a microplate reader (Varioskan® Flash, Thermo, Finland). Inoculated apple juice without antimicrobials was used as control. Absorbance of the well contents was determined at 600 nm. The averages of absorbance values versus time were plotted to form growth curves. The MIC was defined as the lowest concentration required for the inhibition. Experimental growth data were fitted to the Baranyi growth model [18] (link) in DMFit Version 2.1 software (Institute of Food Research, Norwick, UK) to estimate the growth parameters at non-inhibitory doses.

Molva C, & Baysal A.H. (2017). Modeling growth of Alicyclobacillus acidoterrestris DSM 3922 type strain vegetative cells in the apple juice with nisin and lysozyme. AIMS Microbiology, 3(2), 315-322.

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Antimicrobial Efficacy of Oregano Essential Oil

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Oregano essential oil (OEO)
was purchased from Lionel Hitchens Ltd. (Barton Stacey, Hampshire,
UK). Mueller-Hinton Broth (MHB), Mueller-Hinton Agar (MHA), and Maximum
Recovery Diluent (MRD) were purchased from Oxoid (Basingstoke, UK). Escherichia coli (E. coli) (NCIMB 11943), Bacillus cereus (B. cereus) (NCIMB 9373), Staphylococcus
aureus (S. aureus)
(NCIMB 13062), and Pseudomonas fluorescens (P. fluorescens) (NCIMB 9046) were
maintained on Tryptic Soy Agar slants until use at 4 °C. The
siliceous SBA-15 mesoporous material was purchased from Glantreo,
Ireland. Blanket Si substrates were purchased from Sil’tronix,
France. Sulfuric acid (ACS reagent, 95–98%), 3-aminopropyltriethoxysilane
(99%) (APTES), 3-glycidyloxypropyltrimethoxysilane (>98%) (GPTS),
hydrogen peroxide solution (30%), 2-propanol (CHROMASOLV, for high-performance
liquid chromatography (HPLC), 99.9%), and dimethyl sulfoxide (DMSO)
(anhydrous 99.9%) were all purchased from Sigma-Aldrich, Ireland.
Deionized water was purchased from Acros Organics and was used as
necessary.

Sullivan D.J., O’Mahony T.F., Cruz-Romero M.C., Cummins E., Kerry J.P, & Morris M.A. (2021). The Use of Porous Silica Particles as Carriers for a Smart Delivery of Antimicrobial Essential Oils in Food Applications. ACS Omega, 6(45), 30376-30385.

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Listeria monocytogenes inhibition by PHA-based BNPs

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TSB was inoculated with approximately 1 × 10⁷ CFU/mL of L. monocytogenes strain 473, and 0.25 mg/mL of either PHA_lysin293_BNPs, PHA_amidase293_BNPs or the control PHA_BNPs was added for a total reaction volume of 200 μL. Samples were incubated at 37 °C and plated at 30 min intervals for up to 3 h on Listeria Chromogenic agar (Harlequin, Lancashire, UK). A total volume of 100 μL was taken and serially diluted, using Maximum Recovery Diluent (Oxoid Ltd., Basingstoke, UK), to a dilution of 10⁻⁸.The plates were incubated at 37 °C for 48 h. To assess the inhibitory nature of the beads at a lower starting cell number, TSB was inoculated with approximately 1 × 10³ CFU/mL of L. monocytogenes strain 473, and 0.25 mg/mL of either PHA_lysin293_BNPs, PHA_amidase293_BNPs or the control PHA_BNPs was added for a total reaction volume of 200 μL. Samples were incubated at 22 °C and plated at 30 min intervals over a 3 h period onto Listeria Chromogenic agar (Neogen, Lancashire, UK). The plates were incubated at 37 °C for 48 h. The percentage inhibition was calculated using CFU/mL data.

Stone E., Pennone V., Reilly K., Grant I.R., Campbell K., Altermann E, & McAuliffe O. (2022). Inhibition of Listeria monocytogenes by Phage Lytic Enzymes Displayed on Tailored Bionanoparticles. Foods, 11(6), 854.

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Measuring Bedding pH with pH Meter

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The pH value of materials was measured with a pH meter Lab 850 (SiAnalytics GmbH, Mainz, Germany). Its probe was inserted into 25 g of the bedding suspended in 300 mL of Maximum Recovery Diluent (Oxoid, Hampshire, UK). Next, the pH value was read.

Gontar Ł., Sitarek-Andrzejczyk M., Kochański M., Buła M., Drutowska A., Zych D, & Markiewicz J. (2022). Dynamics and Diversity of Microbial Contamination in Poultry Bedding Materials Containing Parts of Medicinal Plants. Materials, 15(4), 1290.

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pH Measurement of Suspended Material

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The pH value was measured with a pH meter Lab 850 (SiAnalytics GmbH, Mainz, Germany). A pH meter probe was inserted into 25 g of the material suspended in 300 mL of Maximum Recovery Diluent (Oxoid, Hampshire, UK) and the pH value was read.

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