METHODS AND COMPOSITIONS FOR PRESERVING BACTERIA
The disclosure provides methods and compositions for the preservation of bacteria. Aspects of the present disclosure provide methods of preparing a preserved bacterial composition comprising flash freezing a bacterial composition and lyophilizing the flash frozen bacterial composition to produce a preserved bacterial composition. In some embodiments, the bacterial composition comprises one or more bacterial strains. In some embodiments, the one or more bacterial strains comprise one or more anaerobic bacterial strains. In some embodiments, the anaerobic bacterial strains are strict anaerobic bacteria.
1 . A method of preparing a preserved bacterial composition, comprising
flash freezing a bacterial composition, and
lyophilizing the flash frozen bacterial composition to produce a preserved bacterial composition.
2 . The method of claim 1 , wherein the bacterial composition comprises one or more bacterial strains.
3 . The method of claim 1 or 2 , wherein the bacterial composition comprises one or more anaerobic bacterial strains.
4 . The method of claim 3 , wherein the anaerobic bacterial strains are strict anaerobic bacteria.
5 . The method of any one of claims 1 - 4 , wherein the bacterial composition comprises one or more bacterial strains belong to the class Clostridia.
6 . The method of claim 5 , wherein one or more bacterial strains belong to the family Clostridiaceae.
7 . The method of claim 6 , wherein the bacteria comprise one or more bacterial strains belonging to the genus Clostridium.
8 . The method of any one of claims 1 - 7 , wherein the bacterial composition comprises one or more bacterial strains selected from the group consisting of Clostridium bolteae, Anaerotruncus colihominis, Sellimonas intestinalis, Clostridium symbiosum, Blautia producta, Dorea longicatena, Clostridium innocuum , and Flavinofractor plautii.
9 . The method of any one of claims 1 - 8 , wherein the bacterial composition comprises one or more bacterial strains comprising 16S rDNA sequences having at least 97% sequence identity with the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1-8.
10 . The method of any one of claims 1 - 9 , further comprising washing the bacterial composition and resuspending the bacterial composition in a formulation buffer.
11 . The method of any one of claims 1 - 10 , wherein the flash freezing is performed by contacting the bacterial composition with a super-cooled surface.
12 . The method of any one of claims 1 - 10 , wherein the flash freezing is performed by contacting the bacterial composition with liquid nitrogen.
13 . The method of any one of claims 1 - 12 , wherein the bacterial composition has a symmetrical shape.
14 . The method of claim 13 , wherein the symmetrical shape is a symmetrical frozen droplet.
15 . The method of any one of claims 1 - 14 , further comprising subjecting the preserved bacterial composition to a temperature of −80° C.
16 . The method of any one of claims 1 - 15 , wherein the lyophilizing comprises a primary drying step and a secondary drying step.
17 . The method of claim 16 , wherein the primary drying step comprises subjecting the flash frozen bacterial composition to a temperature of −10° C. and under a pressure of 70 mTorr.
18 . The method of claim 16 or 17 , wherein the secondary drying step comprises subjecting the flash frozen bacterial composition to a temperature of +20° C. and under a pressure of 70 mTorr.
19 . The method of any one of claims 1 - 18 , further comprising culturing the bacterial composition.
20 . The method of any one of claims 1 - 19 , further comprising determining a level of viability in the preserved bacterial composition after lyophilizing.
21 . The method of claim 20 , wherein the level of viability in the preserved bacterial composition is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% of colony forming units of the bacteria over a period of time.
22 . The method of claim 21 , wherein the period of time is at least 1 week, at least 2 weeks, at least 4 weeks, at least 2 months, at least 3 months, at least 6 months, or at least 1 year or more.