Due　to　the　hindrance　of　flocculated　polymers　and　bacterial　cell　wall,　the　production　of　Bacillus　subtilis　using　monosodium　glutamate　byproduct　(MSGB)　was　low.　With　the　assistance　of　scanning　electron　microscope　images,　effects　of　alkali,　lysozyme,　papain,　ultrasound,　and　their　combinations　on　MSGB　were　evaluated　using　the　results　of　soluble　protein,　carbohydrate,　monosaccharides　and　peptidoglycans.　Alkali　could　dissolve　flocculated　polymers　increasing　21%　soluble　MSGB,　and　thus　enhanced　the　subsequent　treatments　(ultrasound,　lysozyme,　or　papain)　to　increase　14–17%　soluble　MSGB.　As　ultrasound　mainly　released　intercellular　components　(mannose,　and　glucose)　while　lysozyme　or　papain　mainly　released　cell　wall　components　(peptidoglycans),　the　combination　of　alkali,　ultrasound,　and　enzymes　led　to　a　highest　soluble　MSGB　(78%),　yielding　a　maximal　B.　subtilis　production　of　6.6　×　109　colony-forming　units　mL−1.　This　yield　was　about　33　times　that　of　using　untreated　MSGB,　and　the　key　to　improve　B.　subtilis　production　was　the　release　of　carbohydrate.　©　2021　Elsevier　Ltd