Sodium chloride (nacl)

1(w/v)% hair keratin was dissolved in the reaction solution composed of 50 mM HEPES (Gibco, 15630-080), 50 mM NaCl (JUNSEI CHEMICAL, 19015-1250), 0.1% CHAPS (Sigma-Aldrich, C3023), 10 mM EDTA (Sigma-Aldrich, 03609), 5% glycerol (SAMCHUN CHEMICALS, G0274) and 10 mM DTT (Sigma-Aldrich, 43815) at pH 7.2. 5U/ml Casase-3 (Enzo, ALX-201-059) or 5U/ml Casase-6 (Enzo, ALX-201-060) was added to the reaction solution containing hair keratin and incubated at 37 °C for 0, 1, 3, and 24 h. After the reaction, samples were denatured at 70 °C for 10 min in LDS sample buffer (Invitrogen, B0007). Equal amounts of denatured samples were loaded in pre-casted 4–12% Bis-Tris Plus Gels (Invitrogen, NW04120BOX), and the electrophoresis was done by running at 200 V for 22 min. The gel was rinsed three times with distilled water for 5 min each and stained by SimplyBlue SafeStain (Invitrogen, LC6060). After 1 hr of staining, the gel was rinsed using distilled water until the background was removed thoroughly, and then images of the gel was obtained using a commercialized scanner (Canon, TS8090).

IEC was measured by titration. Prepared membranes using all the ionomer dispersions were immersed in 1.0 M HCl to convert exchangeable groups into protons for 24 h. The pretreated membranes were soaked in 1.0 M NaCl (99.5%, JUNSEI Chemical Co., Tokyo, Japan) for 24 h to exchange protons into sodium. The proton in the NaCl solution was titrated by using a titration instrument (848 Titrino plus, Metrohm, Herisau, Switzerland) with a 0.01 M NaOH (99.9%, JUNSEI Chemical Co., Tokyo, Japan) solution [33 (link)]. The IEC was calculated as: $$\mathrm{IEC}\left(\frac{\mathrm{mol}}{\mathrm{g}}\right)=\frac{{\mathrm{C}}_{\mathrm{<a\; class="product"\; href="/product/FyviCZIBPBHhf-iFVQzZ/"\; target="\_blank">NaOH</a>}}\times \left({\mathrm{V}}_{\mathrm{eq}}-{\mathrm{V}}_{\mathrm{blank}}\right)}{{\mathrm{W}}_0}$$
where W₀ is the dry weight of the membranes, C_NaOH is the concentration of a NaOH solution, V_eq is the titrated volume of a NaOH solution for samples, and V_blank is the titrated volume of a NaOH solution for blanks.

The commercial prebiotics used in this study were fructooligosaccharides (FOS) (95.38% purity, Samyang Co., Seoul, Korea), galactooligosaccharides (GOS) (≥98.99% purity, Genofocus Co., Daejeon, Korea), xylooligosaccharides (XOS) (≥95.20% purity, Desang Co., Incheon, Korea), inulin (IN) (≥90% purity, Frutafit HD, Sensus, Netherlands), and 2’-fucosyllactose (2FL) (≥94% purity, APTechnology Corp., Suwon, Korea) of food grade. All other chemicals used in this study were of analytical grade. Vitamin K₁ was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Water peptone and yeast extract were purchased from BD Biosciences (Franklin Lakes, NJ, USA). CaCl₂ ∙ 2H₂O, K₂HPO₄, KH₂PO₄, and NaCl were purchased from Junsei (Tokyo, Japan). Bile salts, L-cysteine hydrochloride, hemin, MgSO₄ ∙ 7H₂O, and NaHCO₃ were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween 80 was purchased from VWR (Radnor, PA, USA).

Kaempferol, myricetin, quercetin, dimethyl sulfoxide (DMSO), MgSO₄·7H₂O, bile salts, l-cysteine hydrochloride, hemin, resazurin solution, pepsin (from porcine mucosa), pancreatin (from porcine pancreas), and formic acid were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). HCl, NaCl, K₂HPO₄, KH₂PO₄, NaHCO₃, MgCl₂, KCl, and CaCl₂·2H₂O were purchased from Junsei Chemical Co., Ltd. (Tokyo, Japan). Tween 80 was purchased from VWR International, LLC (Radnor, PA, USA). Methanol (HPLC grade) and acetonitrile (HPLC grade) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Water (HPLC grade) was purchased from Burdick & Jackson (Muskegon, MI, USA). The fresh porcine small intestine was purchased from a local food market. All other chemicals were of analytical grade or higher.

5-Dimethyl-1-pyrroline-N-oxide (DMPO), FeSO₄, and H₂O₂ were purchased from Sigma Chemical Co. (St. Louis, MO, USA). KH₂PO₄, KCl and NaCl were purchased from Junsei (Tokyo, Japan). Saccharose, dextrose, urea, MgSO₄, MnSO₄, and ZnSO₄ were purchased from Deajung (Siheung, Korea). Peptone G was purchased from Acumedia (Lansing, MI, USA). The grape skin- derived anthocyanins were purchased from Kitolife Co. Ltd. (Pyeongtaek, Korea).