Flavourzyme

Build a system in which 12% sheep bone powder was firstly hydrolyzed by 4.25% alcalase
(activity≥200 U/mg, Solarbio) at pH 9 and 45℃ for 400 min followed by adding
3.65% flavourzyme (activity≥20 U/mg, Solarbio) to the resulted mixture (adjusted to pH
6) and incubating at 55℃ for 240 min. The final mixture was autoclaved at 121℃
for 20 min. L. helveticus were gradually tamed by culturing in MRS broth with
increasing proportion of SBH (supplemented with 1% glucose) from 10% to 100%. The finally tamed
L. helveticus independent of MRS broth was used as the starter in the
following single-factor tests and responsive surface design tests.

Yak bones (leg bone) were obtained from the Tibet Academy of Agricultural and Animal Husbandry Sciences (Tibet, China). The bones were mechanically ground into granules with a diameter ranging from 3–5 mm and stored at −80 °C until analyzed. Pepsin (EC number 3.4.23.1, 1200 U mg⁻¹), trypsin (EC number 3.4.21.4, 92 383 U mg⁻¹), Alcalase (EC number 3.4.21.63, 221 756 U mg⁻¹), Flavourzyme (EC number 3.4.11.1, 15 311 U mg⁻¹), Protamex (56 084 U mg⁻¹), Neutrase (EC number 3.4.24.28, 19 267 U mg⁻¹) and papain (EC number 3.4.22.2, 37 018 U mg⁻¹) were purchased from Solarbio (Beijing, China). Soluble peptides content was measured using a bicinchoninic acid (BCA) Protein Assay Kit from Beyotime (Shanghai, China). Trifluoroacetic acid (TFA), sodium dodecyl sulfate (SDS), and trinitrobenzene sulfonic acid (TNBS) were purchased from Sigma-Aldrich (Shanghai, China). HPLC grade acetonitrile (ACN) was purchased from Thermo Fisher Scientific (Waltham, USA). GP-16 and GD-18 (purity > 98%) was chemical synthesized and purified using high performance liquid chromatography (HPLC) by Beijing Protein Innovation (Beijing, China). All other chemicals and reagents were of analytical grade, unless otherwise stated.