Proteomic response of Saccharomyces boulardii to simulated gastrointestinal conditions and encapsulation

Abstract

Probiotics are live microorganisms conferring health benefits when administered in adequate amounts. However, the passage through the gastrointestinal tract represents a challenge due to pH variations, proteases, and bile salts. This study aimed to evaluate the proteomic response of Saccharomyces boulardii to simulated gastrointestinal digestion and the influence of encapsulation on yeast viability. Different pH values and time periods simulating the passage through different sections of the gastrointestinal tract were applied to unencapsulated and encapsulated yeasts. Encapsulation in 0.5% calcium alginate did not improve yeast survival or induce changes in protein patterns whereas protein extracts from control and digested yeasts showed remarkable differences when separated by SDS-PAGE. Protein bands were analyzed by tandem mass spectrometry. Protein identification revealed unique proteins that changed acutely in abundance after simulated digestion. Carbohydrate metabolism, protein processing, and oxide-reduction were the biological processes most affected by simulated gastrointestinal digestion in S. boulardii.

Nanoproteomic Approach for Isolation and Identification of Potential Biomarkers in Human Urine from Adults with Normal Weight, Overweight and Obesity

“…Tryptic peptides were separated in a reverse phase ultra-performance liquid chromatography using a 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA). Analytical column used was a ZORBAX SB-C18 (100 × 2.1 mm, 1.8 µm, Agilent Technologies, Santa Clara, CA, USA) equilibrated with 1% ACN and 0.1% formic acid (FA) maintained at 35 °C [ 22 , 52 ] with a flow rate of 400 µL/min.…”

“…Peptides were then separated with the following chromatographic conditions: 1 min with 99% of solution A (H2O with 0.1% FA) and 1% of solution B (ACN with 0.1% FA), followed by two linear gradients until reaching 40% of B in 60 min and then 90% of B in 20 min (from 60 to 80 min) and a final gradient of 1 min until reaching 1% of B, with an equilibrium period with 1% of B for 5 min between runs. Peptides eluted from the column were ionized by electrospray with a Dual AJS ESI ionization source applying 3.5 kV, and ions were then analyzed by MS/MS in data-dependent acquisition mode in a 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-ToF) LC/MS System (Agilent Technologies, Santa Clara, CA, USA) and the conditions reported by Morales-Amparano [ 52 ] with a brief change: data were acquired in MS mode at 4 spectra/s scan rate, whereas for MS/MS scan the rate was set at 1 spectrum/s and a maximum of 5 precursors per MS cycle were selected for further peptide fragmentation by collision-induced dissociation.…”

“…Estimated 30 µg of total protein from each sample were reconstituted in 1 M urea, reduced with 10 mM DTT, alkylated with 50 mM iodoacetamide and digested with sequencing grade trypsin (Promega, Madison, WI, USA) overnight at 37 • C. Tryptic peptides were separated in a reverse phase ultra-performance liquid chromatography using a 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, USA). Analytical column used was a ZORBAX SB-C18 (100 × 2.1 mm, 1.8 µm, Agilent Technologies, Santa Clara, CA, USA) equilibrated with 1% ACN and 0.1% formic acid (FA) maintained at 35 • C [22,52] with a flow rate of 400 µL/min.…”

Proteomic identification of allergenic proteins in red oak (Quercus rubra) pollen

“…Protein digestion was carried out overnight at 37 C with sequencing grade trypsin (Promega, Madison, WI, USA). Tryptic peptide separation was performed using the 1290 Infinity LC System (Agilent Technologies, Santa Clara, California, USA) equipped with an analytical column ZORBAX 300SB-C8 (5 mm  2.1 mm x 150 mm, Agilent Technologies), and MS/MS analysis was performed by a 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS system (Agilent Technologies) as previously reported by Morales-Amparano et al 17 Protein identification MS/MS raw data files (.d files) were processed in the Spectrum Mill MS Proteomics Workbench server (Agilent Technologies) to obtain .mzXML files which were transformed into .mgf files in the MSConvert program (available at http://proteowizard. sourceforge.net/).…”

Coated alginate–chitosan particles to improve the stability of probiotic yeast

“…Graff et al (2008) found greater yeast protection when encapsulated using sodium alginate, increasing cell viability in the colon. Morales-Amparano et al (2019) conducted a study of the proteomic response of S. boulardii encapsulated in alginate and found that protection with calcium alginate (0.5%) did not increase the survival of the strain after in vitro digestion.…”