Trichloroacetic Acid

Leiker bAL2²² was used to cross-link the E. coli MG1655 whole-cell lysates. MG1655 was cultured in unlabeled or ¹⁵N-labeled M9 medium and collected at OD₆₀₀ 0.6–0.8. Pellets of 40 OD*ml E. coli cells was resuspended in 0.4 ml lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl). Cell lysates were prepared using a FastPrep homogenizer (6.5 m/s, 20 s, repeat 5 times). After measuring protein concentration, 1 mg of unlabeled and ¹⁵N-labeled lysate was cross-linked separately with 0.33 mg [d0]-bAL2 for 0.5 h at room temperature. Cross-linking reactions were quenched with 20 mM ammonium bicarbonate. The two reactions were mixed together and then precipitated by trichloroacetic acid (TCA) followed by Trypsin digestion. After filtering with a 50 kDa cutoff Amicon Ultra-0.5 Centrifugal Filter Unit, the digested peptides were brought to a volume of 3 mL with 2% ACN, 20 mM HEPES, pH 8.2; the pH was adjusted to 10.0 with ammonia. High-pH reverse-phase separation was used for fractionation. The peptides were eluted with buffer B (80% ACN, 5 mM NH₄COOH, pH 10) gradient. A total of 39 two-minute fractions were collected and then combined into five fractions of similar shades of color (bAL2-linked peptides are bright yellow before cleavage of the biotin tag). Each pooled sample was evaporated to 200–300 µl before enrichment of bAL2-linked peptides on 50 µl high-capacity streptavidin beads. After release of beads and desalting through a C18 column, the five fractions of bAL2-linked peptides were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a Q-Exactive HF mass spectrometer coupled with an EASY-nLC 1000 system (both from Thermo Fisher Scientific). Precursors of the + 1, + 2, + 8, or above, or unassigned charge states were rejected and dynamic exclusion was set to 20 s.

T. reesei RUT-C30 (ATCC 56765) was purchased from ATCC (Manassas, VA) and T. atroviride Ta13 (MUT 6701) was isolated from
wheat seeds (Algeria) and deposited at the Mycotheca Universitatis
Taurinensis (MUT, Turin, Italy).
2,6-Dimethoxyphenol, 3,5-dinitrosalicylic
acid, 4-nitrophenyl butyrate, acetonitrile, bovine serum albumin,
carboxymethyl cellulose, citrate solution, citric acid, formic acid,
malic acid, methanol, Na acetate buffer, Na phosphate buffer, Na phosphate–citrate,
potato dextrose agar, trichloroacetic acid, Tris-HCl buffer, and Triton
X-100 were purchased from Merck (Darmstadt, Germany).

For EPS extraction, 500 mL of commercial UHT low fat milk (La Serenisima, Mastellone Hnos S.A, Argentina) were individually inoculated with fresh pure cultures (1% v/v) and incubated at 20°C, 30°C or 37°C until the acid gels were formed. The fermented milks obtained were heated for 30 min at 100°C in order to favor the detachment and dissolution of the polysaccharide bound to the cells and allow enzymes denaturalization. Trichloroacetic acid 8% w/v (Ciccarelli, Argentina) was added to precipitate the proteins. Then, the samples were centrifuged at 10,000 g for 20 min at 20°C in an Avanti J25 centrifuge (Beckman Coulter Inc., USA) and two volumes of cold ethanol were added to the supernatant obtained. The samples were placed at-20°C overnight and centrifuged at 10,000 g for 20 min at 4°C. EPS pellets were dissolved in hot distilled water and dialyzed against bi-distilled water through a 1 kDa cut-off dialysis membrane (Spectra/Por, The Spectrum Companies, Gardena, CA) for 48 h at 4°C. Finally, the samples were lyophilized. EPS extraction was performed from two independent cultures. The absence of other sugars was determined by thin-layer chromatography and the absence of proteins was evaluated by the Bradford method (Rimada and Abraham, 2003 (link)). EPS amount was estimated by weight of crude EPS lyophilized and expressed as mg/L.

To determine the degree of lipid oxidation, the TBARS of sausage samples was quantified. Shaking for 30 min, a 10-g minced sausage sample was mixed with 50 mL of 7.5% trichloroacetic acid (containing 0.1% ethylenediaminetetraacetic acid). Following that, 5 mL of the supernatant was filtered and mixed with 5 mL of 0.02 mol/L thiobarbituric acid solution at 90 °C for 40 min. 5 mL of chloroform was added after the mixed solution had cooled. A multifunctional microplate reader was used to measure absorbance at 532 and 600 nm (BioTek Epoch, Vermont, USA). The following equation was used to calculate the TBARS value: $$\mathrm{TBARS}(\mathrm{mg}/100\mathrm{g})=\frac{\mathrm{A}532-\mathrm{A}600}{155}\times \left(\frac{1}{10}\right)\times 72.6\times 100.$$ Here, A532 and A600 are the absorbances (532 and 600 nm) of the assay solution.