For determination of nitrogen, carbon, mineral elements, nitrate, as well as the concentration of poly-phenolic compounds, four replicates per treatment were used. Each replicate included a mix of three lettuce heads, which were randomly sampled. Afterwards, the 12 lettuce heads per treatment were quartered. Three quarters of different lettuce heads were pooled as one sample so that all mixed samples contained different lettuce heads. The samples were shock-frozen with liquid nitrogen and kept at -20°C until they were freeze-dried for 48 h (Christ Alpha 1–4, Christ; Osterode, Germany) and ground to a fine powder. Each freeze-dried sample was divided into four sub-samples, one for elemental analysis (EA), one for inductively coupled plasma-optical emission spectrometry (ICP-OES), one for nitrate analysis, and one for high performance liquid chromatography (HPLC).
Christ alpha 1 4
Manufactured by Martin Christ
Sourced in Germany
The Christ Alpha 1-4 is a laboratory centrifuge designed for general-purpose applications. It features a brushless motor, electronic speed control, and a sturdy, high-quality construction. The centrifuge can accommodate a variety of rotor sizes and configurations to suit different sample processing needs.
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13 protocols using christ alpha 1 4
1
Comprehensive Analysis of Lettuce Composition
2
Synthesis of PCEC Copolymer
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PCEC copolymer was prepared by ring-opening polymerization of ε-caprolactone (ε-CL) using Sn (Oct)2 as the catalyst. In summary, ε-CL (7.4 g), PEG (Mn = 6000, 0.74 g), and Sn (Oct)2 (1 wt%) were added to the reaction vessel under the dry nitrogen atmosphere and continued to stir at 130 °C for 7 h. The obtained polymer was dissolved in dichloromethane (DCM) and reprecipitated in a large amount of cold diethyl ether for purification. The resulting polymer was lyophilized with a freeze dryer (model Christ Alpha 1–4 (USA)) and stored at 4 °C for future use.
3
Isolation and Characterization of Plant DNA
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Leaf samples from 1-month-old seedlings of each of the 460 plants used for phenotypic evaluation were freeze dried for 72 h using a freeze dryer (Christ Alpha 1-4, GmbH, Germany). The genomic DNA was isolated using the method described by Orabi et al. (2014) (link) with slight modifications where, DNA was fished out using an inoculation loop followed by washing with 75% ethanol. Then pellets were air dried and suspended in 100 μl of TE buffer pH 8 (10 mM Tris.HCl and 1 mM EDTA) and allowed to dissolve at 4°C for a week. Concentration of the extracted DNA was measured with a nanodrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Wilmington, United States) and the purity and integrity of the DNA was tested using 1% agarose gel electrophoresis.
4
Comprehensive Analysis of Compost Composition
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Ammonium-N content was extracted from 20 g of fresh samples by shaking for 24 h with 100 mL of 0.5 M KCl and centrifuging. The measurement was made using a continuous flow colourimeter (AutoAnalyzer 3; Bran+Luebbe, Le Mans, France). Approximately 1 kg of each composite sample was lyophilised (Christ Alpha 1–4, Christ, Osterode, Germany) for chemical analyses. Sub-samples ground to <0.2mm were used to determine TC, N, P and K. Total C and N were determined using an elemental analyser (NC 2100Soil; CE Instruments, Milan, Italy). Phosphorus and K were determined after mineralization and digestion with HCl. Phosphorus was measured colorimetrically using a spectrophotometer (Cary 100; Varian Inc., Grenoble, France). Potassium was measured by atomic absorption spectrophotometry (AAFS 240; Varian Inc., Grenoble, France). Sub-samples ground to <2mm were used to determine the pH (compost: water 1: 10 w/v) and LIG content [19 ]. As proposed by some authors, in this study we considered the LIG content to be an index of compost stability [15 (link)]. All measurements were performed in duplicate.
5
Biometric Analysis of Plant Growth and Yield
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At harvest (26 days), eight plants per replicate were cut at root collar and sampled to determine biometric parameters and yield. Freshly sampled plants were weighed for total fresh and leaf weight measurements (g plant−1). The height (cm) and number of leaves per plant were then determined. The collected samples were dried in a ventilated oven at 60 °C until constant weight (about three days) for the measurement of the dry weight (g plant−1) and the percentage of dry matter. The dried plant material was then finely ground using an MF10.1 cutting head mill (IKA®, Staufen im Breisgau, Baden-Württemberg, Germany) for the measurement of the mineral concentration. Four plants per replicate were sampled and immersed in liquid nitrogen, stored at −80 °C, and subjected to a freeze-drying cycle (Christ, Alpha 1–4 (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) for the measurement of antioxidant activity, carotenoids, and phenolic acids, while another part was stored at −20 °C for the measurement of chlorophyll concentration.
6
Evaluating Antioxidant and Carotenoid Profiles
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The experimental trial lasted 34 days (29 May to 2 July). At harvest (34 days after transplanting, DAT), ten representative plants per replicate were cut at the root collar, avoiding border plants, and separated into leaves and stems. Shoot fresh weight (fw; g plant−1), leaf fw (g plant−1), stem diameter (mm) using a digital caliper (accuracy ± 0.02 mm; RS PRO, Sesto San Giovanni, Milan, Italy). Six plants per replicate were placed in liquid nitrogen, stored at −80 °C, and freeze-dried (Christ, Alpha 1–4, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) for the determination of antioxidant activities and carotenoids. Another part was stored at −20 °C to determine volatile organic compounds (VOCs).
7
Lettuce Yield and Leaf Characteristics
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Yield was determined as fresh mass per lettuce head at the end of the cultivation from 9 plants per substrate. Additionally, leaf number was counted and leaf area (LA) in cm2 per lettuce head was determined using a leaf area meter (Model LI 3100, LAMBDA Inst. Corp; United States) from 6 plants per substrate.
Three plants from each substrate variant and block were mixed to one sample resulting in 3 samples per substrate for further analysis. Dry mass was determined after freeze-drying (Christ Alpha 1–4, Christ; Osterrode, Germany) for 5 days. The dry matter content expressed in % was calculated by the ratio of the dry mass to the fresh mass.
Three plants from each substrate variant and block were mixed to one sample resulting in 3 samples per substrate for further analysis. Dry mass was determined after freeze-drying (Christ Alpha 1–4, Christ; Osterrode, Germany) for 5 days. The dry matter content expressed in % was calculated by the ratio of the dry mass to the fresh mass.
8
Cheese Composition and Free Amino Acids
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In the processed cheese samples, the dry matter (DM) content and the pH-values were determined. DM content was gravimetrically analysed according to ISO 5534 (ISO, 2004 ) by drying the samples at 102 ± 2 °C to constant mass. The pH values were determined at ambient temperature by inserting a glass tip electrode of a calibrated pH-meter (pH Spear, Eutech
Instruments, Oakton, Malaysia) directly into the cheese at three randomly chosen locations.
In the cheese (CDC and WBC), the determination of free amino acid (FAA) content was undertaken in accordance with the process previously performed by Buňková et al. individual FAAs and the content of similar substances (γ-aminobutyric acid, alanine, aspartic acid, asparagine, arginine, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, tyrosine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, valine; results were expressed in g kg -1 ). Additionally, prior to the particular determination each natural cheese was lyophilised (Christ Alpha 1-4, Christ, Osterode, Germany) twice. Furthermore, each lyophilised sample was extracted twice and each extract was loaded on the column in triplicate (n = 12).
Instruments, Oakton, Malaysia) directly into the cheese at three randomly chosen locations.
In the cheese (CDC and WBC), the determination of free amino acid (FAA) content was undertaken in accordance with the process previously performed by Buňková et al. individual FAAs and the content of similar substances (γ-aminobutyric acid, alanine, aspartic acid, asparagine, arginine, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, tyrosine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, valine; results were expressed in g kg -1 ). Additionally, prior to the particular determination each natural cheese was lyophilised (Christ Alpha 1-4, Christ, Osterode, Germany) twice. Furthermore, each lyophilised sample was extracted twice and each extract was loaded on the column in triplicate (n = 12).
9
Freeze-Drying of Lipoplexes with Trehalose and Mannitol
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Freeze-drying excipient solutions were prepared in water with the following concentrations: 3.5% w/v of trehalose, or 5% w/v of trehalose, or 2.5% w/v of trehalose with 1% w/v of mannitol or 4% w/v trehalose with 1% w/v of mannitol. 3.6 ml of excipient solution were added to lipoplexes suspension (400µl, 1.9µM siRNA). The resulting suspension set in a 50 ml tube (Falcon® centrifuge tube, Corning®, New York, USA) was frozen in liquid nitrogen for 10 min and transferred on the rubber valves for connection to the drying chamber of a laboratory scale freeze-dryer (Christ-Alpha 1-4, Christ, Osterode am Harz, Germany) as shown on Figure 1C. Different lyophilisation cycles were performed based on the drying duration, i.e. 20, 24 or 30 h (duration corresponding to the primary and secondary drying). During runs, process parameters were kept constant, i.e. the ice condenser chamber was set at -60°C and the chamber pressure was set at 0.2 mbar. Heat needed for sublimation was supplied from the atmosphere (the runs were performed at room temperature).
Sample temperature was recorded during freeze-drying runs with the Tempris wireless temperature system (IQ Mobil Solutions GmbH, Wolfratshausen, Germany). SEM images of freeze dried powders were taken on a tabletop scanning electron microscope TM3000 (Hitachi, Japan).
Sample temperature was recorded during freeze-drying runs with the Tempris wireless temperature system (IQ Mobil Solutions GmbH, Wolfratshausen, Germany). SEM images of freeze dried powders were taken on a tabletop scanning electron microscope TM3000 (Hitachi, Japan).
10
Cheese Composition and Free Amino Acids
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In the processed cheese samples, the dry matter (DM) content and the pH-values were determined. DM content was gravimetrically analysed according to ISO 5534 (ISO, 2004 ) by drying the samples at 102 ± 2 °C to constant mass. The pH values were determined at ambient temperature by inserting a glass tip electrode of a calibrated pH-meter (pH Spear, Eutech
Instruments, Oakton, Malaysia) directly into the cheese at three randomly chosen locations.
In the cheese (CDC and WBC), the determination of free amino acid (FAA) content was undertaken in accordance with the process previously performed by Buňková et al. individual FAAs and the content of similar substances (γ-aminobutyric acid, alanine, aspartic acid, asparagine, arginine, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, tyrosine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, valine; results were expressed in g kg -1 ). Additionally, prior to the particular determination each natural cheese was lyophilised (Christ Alpha 1-4, Christ, Osterode, Germany) twice. Furthermore, each lyophilised sample was extracted twice and each extract was loaded on the column in triplicate (n = 12).
Instruments, Oakton, Malaysia) directly into the cheese at three randomly chosen locations.
In the cheese (CDC and WBC), the determination of free amino acid (FAA) content was undertaken in accordance with the process previously performed by Buňková et al. individual FAAs and the content of similar substances (γ-aminobutyric acid, alanine, aspartic acid, asparagine, arginine, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, tyrosine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, valine; results were expressed in g kg -1 ). Additionally, prior to the particular determination each natural cheese was lyophilised (Christ Alpha 1-4, Christ, Osterode, Germany) twice. Furthermore, each lyophilised sample was extracted twice and each extract was loaded on the column in triplicate (n = 12).
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