Purification of viscosin was performed as described previously by Bak et al. (2015) (link). Briefly, P. fluorescens SBW25 was cultivated on King's B agar plates (King et al., 1954 (link)) in darkness at 28 °C for 1 day before being transferred to 20 °C and incubated for another 3 days. Colony material was suspended in demineralized water (MilliQ; Millipore) and homogenized by shaking. Cells and supernatant were separated twice by centrifugation at 4700 r.p.m. for 20 min at 4 °C in a Sigma 3-18K centrifuge (Sciquip). The supernatant was acidified to pH 2.0 with 1 M HCl and left overnight on ice for a precipitate to form. The solution including the precipitate was centrifuged for 27 min at 7000 r.p.m. and 4 °C in a Sigma 3-18K centrifuge. The supernatant was discarded and the precipitate was washed four times with MilliQ water at pH 2.0. The precipitate was dissolved in MilliQ water and pH was adjusted to 8.0 with 1 M NaOH to fully dissolve the precipitate. The solution was lyophilized and the purity of the lipopeptide preparations was verified by HPLC. HPLC analysis was carried out using a Waters Alliance series 2695 system and a Waters model 996 photodiode array detector. The procedure was carried out as described previously by Bak et al. (2015) (link). The same HPLC protocol was used for quantification of viscosin produced in liquid cultures of P. fluorescens SBW25.
Sigma 3 18k centrifuge
Manufactured by Merck Group
Sourced in Germany
The Sigma 3-18K centrifuge is a laboratory equipment designed for general-purpose centrifugation applications. It features a maximum speed of 18,000 rpm and can accommodate sample volumes up to 1,500 mL. The centrifuge is compatible with a variety of rotor types and can be used for tasks such as sedimentation, separation, and concentration of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Xub12 digital, by Grant Instruments (3 mentions)
Milli q, by Merck Group (1 mentions)
Alliance series 2695 system, by Waters Corporation (1 mentions)
Model 996 photodiode array detector, by Waters Corporation (1 mentions)
Genesys 10s uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
P8810, by Merck Group (1 mentions)
11 protocols using sigma 3 18k centrifuge
1
Purification and Quantification of Viscosin
2
Comprehensive Clinical Assessment Protocol
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Prior to the measurement of clinical parameters, the date of birth/age and sex of
each participant was recorded. Heart rate, respiration, blood pressure and body
mass index were measured and recorded. Venous blood samples were collected on
the morning after an 8–10 h overnight fast. The blood samples were stored at 4°C
if not used immediately. The serum was collected by centrifugation at 1000g for 5 min at 22°C using a Sigma 3-18K centrifuge (Sigma-Aldrich, St
Louis, MO, USA). The concentration of serum lipids, albumin, creatinine, fasting
blood glucose and uric acid were measured using reagents supplied by Yongchang
(Shanghai, China). FT3, free T4 (FT4) and thyroid stimulating hormone (TSH) were
measured using reagents supplied by Roche Diagnostics GmbH (Mannheim, Germany).
All of the measurements were undertaken using routine methods in a clinical
laboratory and recorded using equipment supplied by Olympus (Tokyo, Japan). A
second venous blood sample was collected and mixed with 1% heparin to produce
plasma. The concentration of plasma haemoglobin and the white blood cell count
were measured using reagents from Baite (Nanchang, China) and recorded using
equipment supplied by Sysmex (Tokyo, Japan).
each participant was recorded. Heart rate, respiration, blood pressure and body
mass index were measured and recorded. Venous blood samples were collected on
the morning after an 8–10 h overnight fast. The blood samples were stored at 4°C
if not used immediately. The serum was collected by centrifugation at 1000
Louis, MO, USA). The concentration of serum lipids, albumin, creatinine, fasting
blood glucose and uric acid were measured using reagents supplied by Yongchang
(Shanghai, China). FT3, free T4 (FT4) and thyroid stimulating hormone (TSH) were
measured using reagents supplied by Roche Diagnostics GmbH (Mannheim, Germany).
All of the measurements were undertaken using routine methods in a clinical
laboratory and recorded using equipment supplied by Olympus (Tokyo, Japan). A
second venous blood sample was collected and mixed with 1% heparin to produce
plasma. The concentration of plasma haemoglobin and the white blood cell count
were measured using reagents from Baite (Nanchang, China) and recorded using
equipment supplied by Sysmex (Tokyo, Japan).
3
Grapefruit Flavonoid Extraction Protocol
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The frozen, raw material was defrosted at room temperature and allowed to warm up to 25 ± 2 °C. A sample of 1 ± 0.05 g of defrosted grapefruit (flavedo, albedo, or segmental parts) was mixed with the solvent (70% ethanol (v/v)) at 1:10 ratio in a 250 mL round-bottom flask and refluxed in a sand bath at 100 ± 2 °C for one hour. The mixture was left to cool down at room temperature, and then centrifuged with Sigma 3-18K centrifuge (Sigma, Osterode am Harz, Germany) for 10 min at RCP 1789× g, followed by the decantation of the supernatant. The extracts were filtered through PVDF syringe filters (pore size 0.22 µm, Frisenette, Knebel, Denmark) prior to HPLC (high-performance liquid chromatography) analysis. All the extraction conditions are displayed in Table 1 .
4
Ultrasound-Assisted Extraction of Isoflavones
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Ultrasound-assisted extraction was performed using an ultrasound bath (frequency 38 kHz) (Grant Instruments™ XUB12 Digital, Cambridge, England). A sample of 0.3 ± 0.001 g of dried and milled flower heads was macerated in 10 mL of solvent. The extraction of isoflavones was performed by employing different extraction conditions: solvent (50% v/v ethanol and purified water) and extraction time (10 or 30 min), with the processing temperature of 40 ± 2 °C (the temperature is regulated automatically by the ultrasonic bath) [25 (link),26 (link)].
Thermal hydrolysis was completed by transferring the extract to a 250 mL round bottom flask. It was refluxed in a sand bath at 100 °C for 1 h. After the procedure, the mixture was left to cool down and then centrifuged with Sigma 3-18K centrifuge (Sigma, Osterode am Harz, Germany) for 10 min at 3382× g, followed by the decantation of the supernatant. The extracts were filtered through PVDF syringe filters (pore size 0.22 μm, Frisenette, Knebel, Denmark) prior to HPLC analysis. Sample preparation conditions are listed inTable 1 .
Thermal hydrolysis was completed by transferring the extract to a 250 mL round bottom flask. It was refluxed in a sand bath at 100 °C for 1 h. After the procedure, the mixture was left to cool down and then centrifuged with Sigma 3-18K centrifuge (Sigma, Osterode am Harz, Germany) for 10 min at 3382× g, followed by the decantation of the supernatant. The extracts were filtered through PVDF syringe filters (pore size 0.22 μm, Frisenette, Knebel, Denmark) prior to HPLC analysis. Sample preparation conditions are listed in
5
Extraction and Thermal Hydrolysis of Citrus Components
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Firstly, we used an ultrasonic bath (frequency of 38 kHz) (Cambridge, UK, Grant Instruments ™ XUB12 Digital) to macerate 1 ± 0.05 g of flavedo, albedo, or segmental parts in 10 mL of 70% v/v ethanol solvent for a duration of 20 min at a temperature of 50 ± 2 °C (the temperature was regulated automatically by the ultrasonic bath). Thermal hydrolysis was completed by transferring the extract to a 250 mL round-bottom flask and refluxing in a sand bath at 100 ± 2 °C for 1 h. After cooling, the mixture was centrifuged for 10 min at 1789× g using a Sigma 3-18K centrifuge (Sigma, Osterode am Harz, Germany), followed by decantation of the supernatant. Before HPLC analysis, the extracts were filtered through PVDF syringe filters (pore size 0.22 m, Frisenette, Knebel, Denmark). The parameters of sample preparation are listed in Table 1 .
6
Ultrasound-assisted Extraction of Citrus Peels
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Ultrasound-assisted extraction was performed using an UP-250 ultrasonic homogenizer (frequency range 19–25 kHz, 250 W, probes amplitude 35 µm). Firstly, 5 ± 0.25 g of samples (albedo, flavedo, or segmental parts) were defrosted at room temperature before being mixed with the 70% ethanol solvent (v/v) at a 1:5 ratio in a 100 mL chemical beaker and extracted for 1, 3, and 5 min at a temperature of from 33.2 to 40 ± 2 °C. Next, the mixture was centrifuged with Sigma 3-18K centrifuge at room temperature (25 ± 5 °C) (Sigma, Osterode am Harz, Germany) for 10 min at RCF 3382× g, followed by the decantation of the supernatant. Then, the extracts were filtered through PVDF syringe filters (pore size 0.22 µm, Frisenette, Knebel, Denmark) before analyzing with HPLC (high-performance liquid chromatography). All the extraction conditions are displayed in Table 1 .
7
Solvent-Free Centrifugation for Human Milk Lipid Separation
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HM lipid separation was performed according to a previously proposed [28 (link)] and later modified [25 (link)] solvent-free centrifugation approach. Here, 30 mL HM sample aliquots were thawed overnight at 4 °C, followed by tempering at room temperature for at least 20 min. Subsequently, samples were centrifuged for 30 min at 17,800× g and 20 °C in a Sigma 3–18 k centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The upper cake was transferred into microtubes and centrifuged for 20 min at 19,300× g at the same temperature. The obtained upper layer, consisting of pure HM lipids, was removed and used for ATR-FTIR and GC-MS measurements.
8
Monitoring F. prausnitzii Survival in Co-culture
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The three F. prausnitzii strains were incubated in M199 TEER medium to monitor their survival. Secondary bacterial cultures in stationary phase were pelleted by centrifugation at 2500× g for 6 min (11180/13190 rotor, Sigma 3-18K centrifuge, Osterode am Harz, Germany) and resuspended in anaerobic media inside the anaerobic workstation. Bacterial number was estimated using a Petroff-Hauser counting chamber and solutions diluted to yield a concentration of 2.4 × 107 bacterial cells/mL. This bacterial density was chosen so that when used in the co-culture experiments described below it resulted in a multiplicity of infection (MOI) of 100 bacterial cells per intestinal epithelial cell. Triplicates of the bacterial solutions were incubated at 37 °C and the optical density at a wavelength of 600 nm (OD600nm) was recorded (Implen OD600 DiluPhotometer with DC10 DiluCell cuvettes; Total Lab Solutions, Auckland, New Zealand) at 2-h intervals over 24 h.
9
Moisture, Protein, Ash, and Fiber Analysis of Manti
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Moisture content of raw materials and manti samples were determined according to AACCI Method no: 44-01 (2010). Protein and ash contents of flour and manti samples were analysed according to AACCI Method no: 46-13.01 and 08-01.01, respectively (2010). Total dietary fibre (TDF), insoluble dietary fibre (IDF), soluble dietary fibre (SDF) contents of raw materials and manti samples were assessed using an enzymaticgravimetric method (Method no: 32-07.01) with the fibre assay kit (Megazyme, Ireland) (AACCI 2010). Phytic acid (PA) was measured according to Vaintraub & Lapteva (1988) . PA in the raw materials and manti samples was extracted at room temperature with a HCl solution (0.6 N) for 2 h. After centrifugation at 18,550 x g for 30 min (Sigma 3-18 K centrifuge, Göttingen, Germany), supernatant was separated, mixed with Wade reagent (0.03% solution of FeCl 3 .6H 2 O containing 0.3% sulfosalicylic acid), vortexed, and centrifuged again (6600 x g for 10 min, Sanyo MSE, UK). The absorbance was measured at a wavelength, λ=500 nm by GENESYS 10S UV-VIS spectrophotometer (Thermo Scientific, U.S.A.). PA concentration was calculated against a curve built by standard PA solutions with different concentrations varying between 0-58.7 µl/ml prepared from phytic acid sodium salt hydrate (P8810, Sigma). The results are expressed on a dry basis (db).
10
Rapid Milk Fat Separation Method
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Milk fat separation was carried out according to the rapid two-step centrifugation method proposed by Feng et al. [32 (link)] and modified by Luna et al. [33 (link)]. Frozen milk samples were thawed overnight at 4 °C and subsequently tempered at room temperature for at least 20 min. Thirty milliliter aliquots were transferred into falcon tubes and centrifuged at 17,800× g for 30 min at 20 °C in a Sigma 3–18k centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The fat-cake layer was transferred into microtubes and centrifuged at 19,300× g for 20 min at the same temperature, resulting in three separate layers. The upper lipid layer was removed and used for FT-IR and gas chromatography/mass spectrometry (GC/MS) measurements.
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