DOI: 10.25345/c56w96n93
title: MassIVE MSV000099049 - Nutritional Potential of Xuta (Jatropha curcas L.) Protein
title: Three Xuta varieties (JPNT1, JPNT2, and JPNT3) harvested in three consecutive years (2022, 2023, and 2024) were analysed. The harvested seeds were sun-dried for 2-3 days, de-hulled immediately, and the kernels dried further on plastic or fibre mats under direct sunlight until the moisture content was &lt;6% was achieved. The kernels were then stored in jute sacks out of direct sunlight. All seeds were sourced from Jatropowers research farms in India, respectively JPNT1 and 2 from A.S. Kulam Village, Coimbatore district, Tamil Nadu, India, and JPNT3 from Illuppunagaram Village, Tiruppur District, Tamil Nadu, India. Afterwards a hydrothermal treatment was applied prior to the digestion by treating the kernels for 40 min at 100% humidity at 125 C in a combi steamer. After treatment, the kernels were left to air-dry at room temperature for 10 min and subsequently cooled at 4 degree C for another 10 min. All kernels (untreated and treated) were ground in a blender and stored at 4 degree C. The samples were further digested according to Brodkorb et al. (2019) using the INFOGEST method: A sample amount corresponding to 0.2 g protein is weighed, based on a total volume of 40 mL. For the oral phase, the sample was mixed with 4 mL ultra-pure water using an Ultra-Turrax(R) at a maximum rotation speed for 30 sec.. The resulting paste-like sample was then diluted with 4 mL simulated salivary fluid (SSF) containing 1 mM CaCl2(H2O)2. Under constant agitation, the pH was adjusted to 7.0 with 1 M NaOH using an automatic titration device. The appropriate amount of human salivary amylase was then added to achieve an activity of 75 U/mL. To achieve a 1x concentration of SSF, the mixture was filled up to 10 mL with water and was incubated for 2 min. at 37 C. A sample of 2 mL each was collected. Afterwards, 6.4 mL of pre-warmed simulated gastric fluid (SGF) was added, which contained a lipase activity of 60 U/mL and a pepsin activity of 2000 U/mL due to the rabbit gastric extract used, and 0.15 mM CaCl2(H2O)2 in the final digestion mixture. The pH value was adjusted to 3.0 using 1 M HCl solution, and the mixtures were filled up to 16 mL with ultra-pure water. The samples were again incubated at 37 C for 2 h with sufficient shaking. At this point, 2 mL samples were withdrawn. For the final intestinal phase, simulated intestinal fluid (SIF) was added to the mixture in a ratio of 1:1 (v/v), which corresponds to a volume of 7.7 mL. After adjusting the pH to 7.0 using 1 M NaOH solution, a final concentration of 10 mM bile salts was added. The samples were then incubated for 30 min at 37 C with constant stirring. Subsequently, CaCl2(H2O)2 was then added to reach a concentration of 0.6 mM in SIF, followed by the corresponding volume of porcine pancreatin to give a final mixture with an activity of 100 U/mL trypsin and 2,000 U/mL lipase. The pH was then adjusted to 7.0 using 1 M NaOH, after which the mixture was filled up to a final volume of 28 mL, and incubated for 2 h at 37 C with constant shaking. Digestion was stopped by adding the 4-(2-aminoethyl) benzensulfonylfluoride (AEBSF) protease inhibitor, reaching a final concentration of 0.05 mM after 120 min of the intestinal phase. All the samples were then immediately snap frozen in liquid nitrogen. After defrosting, the samples were separated into soluble (S) and insoluble (P) fractions by centrifugation at 4500 rpm and 4 C for at least 30 min, with the supernatant being collected, frozen, and immediately freeze-dried. Peptides were separated an Evosep One LC system using a 40 samples per day (40 SPD) Whisper-zoom standardized gradient (32.5 min). Eluting peptides were analyzed using a timsTOF Pro ion mobility spectrometry (IMS) quadrupole time of flight mass spectrometer operated in data-dependent acquisition parallel accumulation with serial fragmentation (DDA-PASEF) mode. Ionization of peptides took place at a temperature of 180 C and a capillary voltage of 1600 V. A blank run was performed between all samples, during which the ion mobility was automatically recalibrated. MS and MS/MS scan range was 100 to 1700 m/z, the ion mobility ranges (expressed as 1/K0) 0.85 to 1.3 Vs/cm2. A polygon filtering was applied in the m/z and ion mobility area to exclude the low m/z of singly charged ions for PASEF precursor selection. Ramp and accumulation time was set to 100 ms. The number of PASEF ramps was set to 4 with a ramp rate of 9.42 Hz and charge minimum of 0 and a maximum of 5. The quadrupole isolation width was set to 2 for m/z = 700 and 3 for m/z = 800. Collision energy was 27 eV for ion mobility (1/K0) 0.85 Vs/cm2 and 45 eV for ion mobility (1/K0) 1.3 V*s/cm2 respectively. Active precursor exclusion was activated with a window of 0.40 minutes. Brodkorb, A., Egger, L., Alminger, M. et al. INFOGEST static in vitro simulation of gastrointestinal food digestion. Nat Protoc 14, 991-1014 (2019). https://doi.org/10.1038/s41596-018-0119-1