METHODS AND SYSTEMS FOR STABILIZATION AND PRESERVATION OF MICROBES
The present disclosure relates to methods of stabilization of microbial compositions comprising combining a population of pre-served microbial cells with at least one water activity scavenger (WAS) to a desired homogeneity level; and packaging and sealing the mixture of preserved microbial cells and the WAS. The present disclosure further relates to the stabilized microbial compositions and uses thereof.
1 . A method comprising:
combining a population of preserved microbial cells with at least one water activity scavenger (WAS) to a desired homogeneity level; and
packaging and sealing the mixture of preserved microbial cells and the WAS.
2 . A method comprising:
preserving a population of microbial cells to provide a population of preserved microbial cells;
harvesting viable microbial cells from the preserved population of microbial cells to provide a population of viable preserved microbial cells;
combining the population of viable preserved microbial cells with at least one water activity scavenger (WAS) to a desired homogeneity level; and
packaging and sealing the mixture of the population of viable preserved microbial cells and the MMWAS.
3 . The method of claim 2 , further comprising:
identifying a target microbe and/or microbe strain; growing the target microbe and/or microbe strain to produce a population of microbial cells; preparing the population of microbial cells for preservation.
4 . The method of claim 1 , wherein the method further comprises mixing the population of preserved microbial cells with at least one diluent.
5 . The method of claim 4 , wherein the at least one diluent includes calcium carbonate.
6 . The method of claim 1 , wherein the WAS is a microporous mineral WAS, a mesoporous mineral WAS, or a macroporous mineral WAS.
7 . The method of claim 1 , wherein the at least one MWAS is selected from a zeolite, an activated clays, a silica gel, calcium oxide, calcium sulfate, a bentonite, sorbitol, calcium chloride, a poly(acrylic acid) sodium salt, sodium chloride, and tamarind seed galactoxyloglucan.
8 . The method of claim 1 , wherein the at least one WAS includes a microporous aluminosilicate mineral.
9 . The method of claim 2 , wherein preserving the population of microbial cells comprises preservation by vaporization (PBV).
10 . The method of claim 1 , wherein the preserved microbial cells are preserved in a glass state.
11 . The method of claim 1 , wherein the preserved microbial cells have a high glass transition temperature.
12 . The method of claim 1 , wherein the at least one WAS is a microporous mineral WAS comprising a porosity percentage of between 20% and 50%.
13 . The method of claim 1 , wherein the at least one WAS is a microporous mineral WAS comprising pores and corner-sharing aluminosilicate tetrahedrons joined into three-dimensional frameworks.
14 . The method of claim 1 , wherein the at least one WAS is a microporous mineral WAS comprising a complex formula of: (Na,K,Ca) 2-3 Al 3 (Al,Si) 2 Si 13 O 36 -12 H 2 O
15 . The method of claim 1 , wherein the at least one WAS comprises a zeolite.
16 . The method of claim 1 , wherein the at least one WAS comprises clinoptilolite zeolite.
17 . The method of claim 1 , wherein the population of preserved microbial cells comprises one or more of a Clostridium spp. bacterium, a Succinivibrio spp. bacterium, a Butyrivibio spp. bacterium, a Bacillus spp. bacterium, a Lactobacillus spp. bacterium, a Prevotella spp. bacterium, a Syntrophococcus spp. bacterium, a Ruminococcus spp. bacterium, a Caecomyces spp. fungus, a Pichia spp. fungus, an Orpinomyces spp. fungus, a Piromyces spp. fungus, or a species of the Lachnospiraceae family.
18 . (canceled)
19 . (canceled)
20 . The method of claim 17 wherein:
a. the Clostridium spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6;
b. the Succinivibrio spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 11;
c. the Pichia spp. comprises an ITS sequence comprising at least 97% sequence identity to SEQ ID NO: 2;
d. the Bacillus spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 4;
e. the Lactobacillus spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9;
f. the Prevotella spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 10; or
g. the species of the Lachnospiraceae family comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 12.
21 . The method of claim 1 , wherein the population of preserved microbial cells comprises a Ruminococcus bovis bacterium, a Succinivibrio dextrinosolvens bacterium, or a Caecomyces spp. fungus.
22 . The method of claim 1 , wherein the population of preserved microbial cells comprises a Clostridium butyricum bacterium, Clostridium butyricum sp. nov., a Clostridium beijerinckii bacterium, a Clostridium beijerinckii sp. nov. bacterium, a Pichia kudriazevii fungus, a Pichia kudriazevii fungus, a Pichia kudriazevii sp. nov. fungus, a Butyrivibio fibrosolvens bacterium, a Ruminococcus bovis bacterium, or a Succinivibrio dextrinosolvens bacterium.
23 . The method of claim 3 , wherein the identifying the target microbe and/or microbe strain comprises: processing of a plurality of samples collected from a sample animal population to identify the one or more target microbes and/or microbe strains, the processing including: for each sample of the plurality of samples: measuring at least one metadata associated with the sample animal population; detecting the presence of a plurality of microorganism types and determining an absolute number of cells of detected microorganism types; determining a relative measure of one or more strains of detected microorganism types of the plurality of microorganism types; determining a set of target microbes and/or microbe strains and respective absolute cell counts based on the absolute number of cells of a detected microorganism type and the relative measure of the one or more microorganism strains for that microorganism type, and filtering by activity level; and analyzing the set of target microbes and/or microbe strains and respective absolute cell counts with the measured metadata to identify relationships between target microbes and/or microbe strains and measured metadata.
24 . The method of claim 1 , wherein the preserved microbial cells are spores.
25 . The method of claim 1 , wherein the preserved microbial cells are vegetative cells.
26 . A product prepared by the methods of claim 1 , comprising a population of preserved microbial cells and a WAS.
27 . The product of claim 26 , wherein the population of preserved microbial cells comprises one or more of a Clostridium spp. bacterium, a Succinivibrio spp. bacterium, a Butyrivibio spp. bacterium, a Bacillus spp. bacterium, a Lactobacillus spp. bacterium, a Prevotella spp. bacterium, a Syntrophococcus spp. bacterium, a Ruminococcus spp. bacterium, a Caecomyces spp. fungus, a Pichia spp. fungus, an Orpinomyces spp. fungus, a Piromyces spp. fungus, or a species of the Lachnospiraceae family.
28 . (canceled)
29 . (canceled)
30 . The product of claim 26 , wherein:
a. the Clostridium spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 6;
b. the Succinivibrio spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 11;
c. the Pichia spp. comprises an ITS sequence comprising at least 97% sequence identity to SEQ ID NO: 2;
d. the Bacillus spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 4;
e. the Lactobacillus spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9;
f. the Prevotella spp. comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 10; or
g. the species of the Lachnospiraceae family comprises a 16S rRNA sequence comprising at least 97% sequence identity to SEQ ID NO: 12.
31 . The product of claim 26 , wherein the population of preserved microbial cells comprises a Ruminococcus bovis bacterium, a Succinivibrio dextrinosolvens bacterium, or a Caecomyces spp. fungus.
32 . The product of claim 26 , wherein the population of preserved microbial cells comprises a Clostridium butyricum bacterium, Clostridium butyricum sp. nov., a Clostridium beijerinckii bacterium, a Clostridium beijerinckii sp. nov. bacterium, a Pichia kudriazevii fungus, a Pichia kudriazevii sp. nov. fungus, a Butyrivibio fibrosolvens bacterium, a Ruminococcus bovis bacterium, or a Succinivibrio dextrinosolvens bacterium