Bile resistant bacillus composition secreting high levels of phytase

A bacillus composition, which comprises from 105 to 1012 CFU/g bacillus spore cells, wherein the bacillus composition is characterized by:

• (i):
• the bacillus spores have a rapid germination and outgrowth from spore to vegetative cell in presence of a bile salt medium comprising 4 and 6 mM bile salts, defined by that the bacillus spores reaches a vegetative cell growth point of 0.4 OD630 within less than 18 and 19 hours, respectively, wherein the vegetative cell growth point is the point in the growth curve where the OD value starts to increase (due to growth of the vegetative cells) in a continuous way and reaches an OD630 of 0.4;
• (I):
• wherein the bile salt medium is the standard known non-selective Veal Infusion Broth (VIB) medium of example 1 herein supplemented with a bile salt mixture comprising the conjugated bile salts taurodeoxycholate and glycodeoxycholate and the deconjugated bile salt deoxycholate in the proportions 60% of the taurodeoxycholate, 30% of the glycodeoxycholate and 10% of deoxycholate;
• and
• wherein the OD assay analysis is performed by the following steps:
• (a):
• filling a well in a microtiter plate with 0.150 ml bile salt medium having 108 bacillus spores per ml medium (i.e. this is time zero);
• and
• (b):
• incubating the plate at 37° C. under atmospheric conditions and measuring the OD630 values, using a spectrophotometer and with agitation before each reading, to get a representative growth curve over time;
• and
• (ii) the bacillus vegetative cells are producing phytase in an amount of at least 1.25 times more than the reference bacillus cell DSM 19467, wherein the produced phytase amount is measured by the standard phytase assay of example 2 herein after 4 hours growth at 37° C. in the standard known non-selective Heart Infusion Broth (HIB) medium of example 2 herein;
• and
• wherein the phytase assay analysis is performed by the following steps:
• (a):
• making a overnight culture of bacillus vegetative cells in a enriched culture medium;
• and
• (b):
• transferring a 1% inoculum from the overnight culture to HIB medium (i.e. this is time zero) and incubation at 37° C. until phytase activity measurement.