The fruits were first rinsed with tap water and freeze-dried (Christ Alpha 1–4 LD plus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) over 72 h, at −42 °C and 0.10 mBar. The freeze-dried fruits were grounded and deposited at 4 °C until analysis (no more than one month).
Christ alpha 1 4 ld plus
Manufactured by Martin Christ
Sourced in Germany
The Christ Alpha 1–4 LD plus is a freeze-drying system designed for laboratory use. It features a condenser with a capacity of 4 liters and can accommodate up to four sample chambers. The product is intended for freeze-drying applications in research and development environments.
Automatically generated - may contain errors
Lab products found in correlation
Rotary evaporator, by Martin Christ (1 mentions)
Whatman no 1 filter paper, by Cytiva (1 mentions)
Almond β glycosidase, by Merck Group (1 mentions)
Agilent vials, by Agilent Technologies (1 mentions)
Speedvac spd 300dda, by Thermo Fisher Scientific (1 mentions)
Disodium hydrogen phosphate, by Merck Group (1 mentions)
Pellet die, by Specac (1 mentions)
15 protocols using christ alpha 1 4 ld plus
1
Freeze-Drying and Grinding of Fruits
2
Extraction of Plant Metabolites
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The powdered plant samples of A. implexa, H. scandens, S. quadrifida and S. glomulifera (0.7 L × 3, 24 h intervals), A. falcata (0.6 L × 3, 24 h intervals), C. glabella and S. glyciphylla (0.5 L × 3, 24 h intervals), and sap of E. haemastoma (0.2 L × 3, 24 h intervals) were each extracted with 70% aqueous ethanol at room temperature with occasional shaking (for plant sample amounts see Table 1 ). The extracts were filtered under vacuum through Whatman filter paper No. 1; then the solvent removed by evaporation using a Buchi rotary evaporator at 38°C before the crude samples were freeze-dried on a CHRIST alpha 1–4 LD plus (UK) freeze dryer. The quantities of the crude extracts obtained are given in Table 1 .
3
Phytochemical Extraction and Characterization
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The plant was rinsed and divided into several fractions; whole plant, flowers, leaves, green stems, and brown stems. The phytochemicals were extracted by maceration and performed over multiple stages where after finely chopping the plant portions, each fraction (50-100 g according to the abundance of each part) was fully submerged in a suitable volume of distilled water and ethanol (100%) in separate light blocking beakers and agitated at ambient temperature for 8-12 h then for the same duration at 37°C. They were then filtered and the process repeated for the obtained marks. Finally, the resulting fractions were concentrated using a rotary evaporator under reduced pressure at 60°C for aqueous and 40°C for ethanolic fractions, and finally, they were frozen at −80°C and freeze-dried using a lyophilizer (Christ Alpha 1-4 LDplus, Martin Christ, Germany) for 2-3 days at −20°C to obtain the powders to study. To assess the obtained powders, they were dissolved in sterile distilled water, and aliquots with a defined concentration were prepared for further analysis.
4
Extraction and Analysis of Raspberry Ketone from N. benthamiana
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N. benthamiana leaves were collected 6 days after infiltration
and immediately frozen in liquid nitrogen. Samples were lyophilized
(CHRIST Alpha 1-4 LD Plus, Martin Christ, Osterode am Harz, Germany)
and powdered with a ball mill grinder (MM301, RETSCH, Haan, Germany)
for 2 × 1 min at 29 Hz. Lyophilized samples were weighed with
a precision scale, and 100 mg of each was suspended in 5 mL of 80%
(v/v) aqueous methanol and sonicated for 20 min, followed by centrifugation
for 15 min at 3000 rpm. Two methanol extractions were performed for
each sample, and supernatants were combined into 50 mL Falcon tubes
and evaporated to dryness (SpeedVac SPD 300DDA, Thermo Scientific,
Bellefonte, USA).
Crude plant extract samples were mixed with
500 μL of 10 mM (MES)-KOH (2-(N-morpholino)
ethanesulfonic acid) and 150 μL of 5 mg/mL almond β-glycosidase
(Sigma-Aldrich, St. Louis, USA) and incubated at 37 °C for 15
h with gentle shaking. Samples were then transferred into glass KIMAX
tubes, and raspberry ketone was extracted with 4 mL of methyl tert-butyl ether (MTBE), transferred into 2 mL Agilent vials,
and evaporated to dryness under nitrogen flow.
and immediately frozen in liquid nitrogen. Samples were lyophilized
(CHRIST Alpha 1-4 LD Plus, Martin Christ, Osterode am Harz, Germany)
and powdered with a ball mill grinder (MM301, RETSCH, Haan, Germany)
for 2 × 1 min at 29 Hz. Lyophilized samples were weighed with
a precision scale, and 100 mg of each was suspended in 5 mL of 80%
(v/v) aqueous methanol and sonicated for 20 min, followed by centrifugation
for 15 min at 3000 rpm. Two methanol extractions were performed for
each sample, and supernatants were combined into 50 mL Falcon tubes
and evaporated to dryness (SpeedVac SPD 300DDA, Thermo Scientific,
Bellefonte, USA).
Crude plant extract samples were mixed with
500 μL of 10 mM (MES)-KOH (2-(N-morpholino)
ethanesulfonic acid) and 150 μL of 5 mg/mL almond β-glycosidase
(Sigma-Aldrich, St. Louis, USA) and incubated at 37 °C for 15
h with gentle shaking. Samples were then transferred into glass KIMAX
tubes, and raspberry ketone was extracted with 4 mL of methyl tert-butyl ether (MTBE), transferred into 2 mL Agilent vials,
and evaporated to dryness under nitrogen flow.
5
Rosehip Powder Extraction Protocol
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Rosehip fruits (Rosa canina L.) were obtained from the local producer (Galați, Romania), harvested in the winter of 2021, at full maturity. The fruits were sorted, washed, crushed, immediately frozen (−18 °C), and subjected to freeze-drying (CHRIST Alpha 1–4 LD plus, Germany) at −42 °C under a pressure of 10 MPa for 48 h. The resulting powder with the diameter of the particle sizes passed through a stainless-steel mesh sieve between 0.2 and 0.4 mm was collected in dark glass containers and stored at 4 °C until extraction. The rosehip powder oil content and the moisture content were 2.67 ± 0.03% (AOAC, 1998 ) and 5.42 ± 0.01%, respectively.
6
Freeze-drying and Redispersion of S/Cur
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Fresh S/Cur was freeze-dried (Christ Alpha 1-4LDplus, Christ, Germany) at −55 ℃ for 48 h. Then the S/Cur powder was dispersed in deionized water under stirring at 600 rpm for 20 min. The appearance of the redispersed S/Cur was observed and further analyzed by the particle size.
7
Protein and ELISA Analysis of Basil
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For performing protein content, SDS-PAGE and ELISA tests, fresh tissue was lyophilized using a Laboratory Freeze Dryer (Christ Alpha 1-4 LDplus), and then stored at room temperature. Basil samples were extracted with Tris-glycine buffer (T-G, pH = 8.3) composed of 0.05 M Tris and 0.33 M glycine. For this purpose, 0.05 g of dried plant tissue was homogenized in 3 ml of T-G buffer and incubated on a laboratory shaker (TTS 2, Yellow Line, IKA -Werke GmbH & Co. KG, Staufen, Germany) for 2 h. Samples were then centrifuged at 4000 rpm for 10 min and the supernatants were transferred into new tubes and stored until analysis at -20°C.
8
Collagen Crosslinking Quantification
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All chemicals (hydrogen peroxide, sodium hydroxide, citric acid, sodium chloride, petroleum ether, acetone, methanol, ethanol, sodium carbonate, Folin–Ciocalteu reagent, and gallic acid) used were of analytical grade and used without further purification. The McIlvaine buffer was prepared at pH = 5 using a mixture of 0.1 M citric acid and 0.2 M disodium hydrogen phosphate (Sigma Aldrich). The buffer prepared was stored at 4 °C for further use. Collagen type I (TH) purchased from Sigma Aldrich was used as a reference. Crosslinker solution was lyophilized in a freeze dryer (Christ Alpha 1–4 LD plus (Martin Christ, Germany)) and microplate determination of crosslinked phenolic compounds was performed using a microplate reader (Tecan Pro 200, Tecan Trading AG, Männedorf, Switzerland).
9
Spectroscopic Analysis of Fen Plant Species
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In summer 2018, 24 shoot samples were collected from plants of three species from the Schlöppnerbrunnen fen, the same site at which the plants for the growing experiment were taken. These species were Carex rostrata, Eriophorum vaginatum and Molinia caerulea Moench. The shoots were frozen and subsequently freeze dried (Christ Alpha 1-4 LDplus, Osterode am Harz, Germany).
The plants were milled according to the procedure described inSection 4.1.4 . Milled samples were subjected to an FTIR analysis (as described in Section 4.1.6 ) and total element mass fractions were determined with X-ray fluorescence spectroscopy at the University of Münster, Germany. For the latter, a wavelength dispersive X-ray fluorescence spectrometer (WD-XRF Rigaku ZSX Primus II, Tokyo, Japan) was used. Therefore, 500 mg of the sample was formed into a pellet at a load of 6–7 t using a pellet die (Specac, Orpington, UK), and pellets were stored in polyethylene bags in a desiccator until measurement.
The plants were milled according to the procedure described in
10
Olive Pomace Processing and Preservation
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Fresh olive pomace was sourced from a local olive processing plant carrying out two-phase olive oil extraction from olives of the Manaki variety cultivated in the Peloponnese region. The raw pomace had a moisture content of 45% w/w on a wet basis and a residual oil content of 7.4% w/w on a dry basis. The pomace was kept at 0 °C until further processing. For experiments where dry material was required, the pomace was either air-dried at 40 °C for 24 h or freeze-dried at −52 °C and 0.080 mbar for 48 h using a Christ Alpha 1–4 LD plus freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Harz, Germany). The dry pomace was stored in sealed polyethylene-polypropylene sachets at room temperature.
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