Increased shelf-stability probiotic strains

Abstract

Evolved strains of the genera Bifidobacterium and Lactobacillus are provided. The evolved strains have probiotic properties and enhanced storage stability under both wet and dry conditions. The strains of the present invention contain significant genetic mutations and phenotypic changes compared to wild-type strains.

Description

[Technical field]

[0001] The present invention relates generally to the field of microbiology and to the evolution of strains of the genera Bifidobacterium and Lactobacillus with enhanced storage stability. [Background technology]

[0002] Increasing awareness of the relationship between diet and health has led to an increased demand for foods that can both enhance health and provide basic nutrition, examples of which include probiotics.

[0003] The term probiotic, meaning "for-life", comes from Greek and already indicates the most essential properties of probiotic cultures. The term probiotic was first used by Lilly and Stillwell, 1965, to describe "a substance secreted by one microorganism that stimulates the growth of another" and was thus contrasted by the term antibiotic (Lilly and Stillwell, 1965). It may be because of this positive and general assertion of the definition that the term probiotic was subsequently used for other objects and acquired a more general meaning. Here, the definition of the term refers to "a live microorganism that beneficially affects the health of the host".

[0004] Various studies have demonstrated that the average human and animal intestine contains approximately 100 different species of bacteria (including "probiotic" bacteria, such as those belonging to the genera Lactococcus, Streptococcus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Lactobacillus, and Bifidobacterium).

[0005] Regular intake of "probiotic" bacteria has been reported to enhance gastrointestinal function and stability, improve protection against infections and carcinogenic mutagens, strengthen the immune system, reduce lactic acid intolerance, improve digestion and nutrient absorption, lower blood cholesterol, and reduce the risk of allergies and wheat intolerance.

[0006] Probiotic bacterial cultures are found today in a wide range of ingestible products, such as dairy products, and various emulsions, such as oil-in-water and water-in-oil products. The incorporation of probiotics into such formulations is particularly challenging due to the harsh processing conditions.

[0007] Probiotic cultures face various stresses during production and subsequent storage. During the production process, probiotics may be exposed to cold, heat, osmotic, oxidative and acidic stresses (Upadrasta et al. 2011). The stresses associated with freeze-drying or spray-drying mainly lead to lethal damage in the cell membrane, but also affect the cell wall, ribosomes and DNA / RNA (Castro et al. 1997; Simpson et al. 2005; Mills et al. 2011). Depending on the storage conditions, it is not uncommon to see a loss of viable cell count (measured as colony forming units (CFU)) of more than 90% over a 2-year storage period.

[0008] To mitigate storage losses, many probiotic manufacturers add more viable cells to the product at the time of production ("overfeed") to ensure that the viable cell count at the end of storage (at the specified storage conditions) still exceeds the product's label claim. Strains with increased storage stability allow the incorporation of fewer viable cells per product at production, and the product still meets or exceeds the storage label claim at the end of storage. Reducing the cell count (i.e., less than "overfeed") in turn leads to a reduction in the fermentation volume required to produce the same amount of product, ultimately resulting in a significant increase in the profitability of the process. Furthermore, in many cases, the application form, e.g., pills, can limit the amount of overfeed that may be applied by not exceeding a certain maximum size. Thus, the feasibility of commercializing a strain is highly dependent on its storage stability to provide the desired viable cell count over the product's lifespan.

[0009] A growing body of evidence suggests that physiological changes induced by pre-storage stresses can improve storage stability, measured as viable cell counts (CFU) after storage. Pre-storage exposure of Lactobacillus rhamnosus (HN001) to stressful levels of heat or salt was significantly correlated with improved storage stability compared to non-stressed control cultures (Prasad et al. 2003). NaCl stress during the late growth phase improved the freeze-drying survival of Lactobacillus bulgaricus, while fermentation of Lactobacillus reuteri at high temperature and pH improved cell survival after freeze-drying (Li et al. 2014, Liu et al. 2014a). Changes in pH and temperature induced modifications in the composition of the cell membrane, which appeared to improve the viability of the cells after freeze-drying (Liu et al. 2014b). Pre-exposure of probiotic bacteria to heat, acid, bile, oxygen, and H2O2 all led to enhanced survival during drying (Upadrasta et al. 2011). Similarly, US Pat. No. 5,399,633 teaches a method to improve the viability of dried Pseudomonas fluorescens, which consists of limiting the nitrogen source during fermentation and then leaving the strain in stationary phase for at least 6 hours. Further improvement was achieved by applying a heat shock before drying.

[0010] The present invention is distinguished from the aforementioned prior art by generating and selecting cells with increased storage stability based on genetic rather than physiological changes, and the mutations underlying this heritable phenotypic improvement can be identified by whole genome sequence analysis.

[0011] US Patent No. 5,999,363 teaches that natural selection by multiple cycles of heat shock treatment was followed by a growth step in mild conditions to isolate more heat shock stable strains. Using the same approach, (Kulkarni et al. 2018) was able to obtain L. acidophilus strains with higher survival rates during heat shock treatment and storage for 7 days. In each of these above-mentioned reports, the strains were exposed to stress for a relatively short period of time. No effort was made to understand the genetic basis of the increased survival rate or to determine whether this characteristic is a stable and heritable phenotype.

[0012] Studies have suggested that there is a genetic basis for linking strains with increased resistance to various stresses and increased storage stability. For example, in a set of various Bifidobacterium species, Simpson et al. observed a correlation between intrinsic resistance to heat or oxygen stress and viability after storage (Simpson et al. 2005). Also, the teachings presented make use of the surprising finding that a population of cells grown simultaneously under successive temperature stresses increases the fraction of cells in the population that exhibit improved storage stability characteristics. These studies are intriguing, but they have not elucidated the genetic changes that the inventors attribute to the improved storage stability.

[0013] Therefore, the problem to be solved is to bring to light the genetic basis of storage stability, since this will provide the industry with a reproducible basis for use in bacterial cultures in general, and those with probiotic specificity in particular, to enhance storage stability and reduce costs associated with dose oversupply.In addition, there is a need to develop selection and screening tools to identify related evolved strains with good manufacturing properties combined with improved storage stability. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] U.S. Patent No. 5,728,574 [Patent Document 2] U.S. Patent No. 8,741,622 Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention addresses the challenges presented by providing experimentally evolved strains of Bifidobacterium and Lactobacillus that demonstrate enhanced storage stability based on genetic, rather than physiological, changes, where the cells grow under conditions of continuous exposure to stress. [Means for solving the problem]

[0016] Thus, the present invention provides a thermally evolved strain of the genus Bifidobacterium, the strain having at least one mutation selected from the group consisting of: a) at least one mutation in the gene encoding polyribonucleotide nucleotidyl transferase; b) at least one mutation in the CheY-like receiver domain; c) at least one mutation in the gene encoding the SSU ribosomal protein; d) at least one mutation in the intergenic region upstream of the gene encoding oleate hydratase and downstream of the gene encoding phosphoglycerate mutase; e) at least one mutation in the gene encoding the Pup ligase PafA' paralog; f) at least one mutation in the gene encoding UTP-glucose-1-phosphate uridylyltransferase; g) at least one mutation in the gene encoding the arginine / ornithine antiporter ArcD; and h) at least one mutation in the gene encoding the pyridoxine biosynthetic glutamine amidotransferase The mutations described in (parts a) to h) are compared to the genomic sequence of the ancestral strain shown in SEQ ID NO: 1, The evolved strains have greater storage stability or effective productivity compared to the ancestral strain (Bi-07, ATCC PTA SD5220).

[0017] In another embodiment, the present invention provides a thermally evolved strain of Bifidobacterium animalis subsp. lactis (BBi 6.6), deposited at the DSMZ under number DSM 33461 on March 4, 2020.

[0018] In another embodiment, the present invention provides a thermally evolved strain of Lactobacillus acidophilus NCFM, the strain having at least one mutation selected from the group consisting of: a) at least one mutation in the gene encoding the 30S ribosomal protein S18; (rpsR); b) at least one mutation in the gene encoding pyridoxal kinase (LBA1007); c) at least one mutation in the gene encoding the ATPase; (clpC); d) at least one mutation in the gene encoding the gnt transcriptional regulator; (gntR); e) at least one mutation in the intergenic region affecting expression of the gene encoding uridine monophosphate kinase; (LAB1268); f) at least one mutation in the gene encoding a cation transporter (LBA0098); and g) at least one mutation in the gene encoding transposase (LBA0127) Mutations (a) to (g) are compared with the genome sequence (SEQ ID NO: 2) of the parent strain (Lactobacillus acidophilus NCFM, ATCC 700396); The evolved strains have greater storage stability compared to the parent strain (Lactobacillus acidophilus NCFM, ATCC 700396).

[0019] In another embodiment, the present invention provides an evolved strain of Lactobacillus acidophilus (NCFM 6.3) deposited at the DSMZ under number DSM 33463 on March 4, 2020.

[0020] In another embodiment, the present invention provides a NaCl evolved strain of Bifidobacterium animalis subsp. lactis, the strain having at least one mutation selected from the group consisting of: a) at least one mutation in the gene encoding class II fructose-bisphosphate aldolase; b) at least one mutation in the gene encoding the first hypothetical protein; c) at least one mutation in a gene encoding a putative ABC transporter integral membrane protein; d) at least one mutation in the gene encoding a KH domain RNA-binding protein (YlqC); e) at least one mutation in a gene encoding a regulator of polyketide synthase; f) at least one mutation in a gene encoding an ABC transporter ATP-binding protein; g) at least one mutation in a gene encoding a putative esterase; h) at least one mutation in the gene encoding a signal transducing histidine kinase; i) at least one mutation in a gene encoding the permease component of an ABC-type amino acid transport system; j) at least one mutation in the gene encoding histidinol-phosphate aminotransferase; k) at least one mutation in the gene encoding the DNA-dependent RNA polymerase beta subunit; l) at least one mutation in a gene encoding a second hypothetical protein; m) at least one mutation in the gene encoding arginyl-tRNA synthetase; and n) at least one mutation in a gene encoding a third hypothetical protein.

[0021] In another embodiment, the present invention provides a NaCl evolved strain of Bifidobacterium animalis subsp. lactis (BBi 2.7), deposited at the DSMZ under number DSM 33462 on March 4, 2020.

[0022] In another embodiment, the present invention provides a salt-evolved strain of Bifidobacterium animalis subsp. lactis, wherein the strain has at least one mutation selected from the group consisting of: a) A mutation in the gene encoding the SSU ribosomal protein S5p (S2e) (the mutation is a C to T change at position 477512); b) a mutation in the intergenic region downstream of the gene encoding phosphoglycerate mutase and upstream of the gene encoding oleate hydratase (the mutation is a C to A change at position 530981); c) a mutation in the gene encoding pantothenate kinase type III (CoaX-like) (the mutation is a G to A change at position 660753); d) a mutation in the gene encoding argininosuccinate synthase (the mutation is a C to T change at position 857663); e) a mutation in the intergenic region downstream of tRNA-Asn-Gtt and upstream of a gene encoding a hypothetical protein (the mutation is a G to A change at position 895939); f) a mutation in the gene encoding a putative glucose uptake permease (the mutation is a G to A change at position 1259521); g) a mutation in the gene encoding the arginine / ornithine antiporter (ArcD) (the mutation is a C to T change at position 1480689); h) A mutation in the gene encoding the pyridoxine biosynthetic glutamine amidotransferase glutaminase subunit (the mutation is a T to C change at position 1639044). The positions given in (parts a) to n) are relative to the ancestral genome sequence (SEQ ID NO: 1).

[0023] In another embodiment, the present invention provides a method for the evolution and identification of probiotic strains exhibiting at least one beneficial phenotype, comprising: a) providing a culture of an ancestral probiotic strain that has the capacity to undergo natural genetic changes leading to increased genetic diversity; b) subjecting the culture of step a) to evolutionary pressure over multiple generations to increase the frequency of mutations linked to beneficial phenotypes compared to an ancestral strain; c) subjecting the culture of step b) to a selection process to isolate evolved strains. (The evolved strains continue to have probiotic efficacy).

[0024] BRIEF DESCRIPTION OF THE FIGURES, BIOLOGICAL DEPOSITS AND SEQUENCE DESCRIPTIONS Applicant has made the following biological deposit for patent procedures under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms with Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7B, 38124 Braunschweig, Germany:

[0025] [Table 1]

[0026] It is required that biological material shall only be made available by delivery of a sample to an expert designated by the claimant. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 illustrates an experimental scheme to test whether selection for growth at elevated temperatures leads to improved storage stability. [Diagram 2] FIG. 2 is a graph showing the percentage of surviving cells per ancestor or evolutionary clone after 22 days of storage at 50° C., based on surviving cells before storage. [Diagram 3] FIG. 3 is a graph showing the effective productivity of cultures BBi Wildtype C1 stored for X=14 d (dotted grey) or X=28 d (solid grey) and BBi Heat 6.6 stored for X=14 d (dark upward diagonal stripes) or X=28 d (light upward diagonal stripes) at 30° C. and 40% relative humidity. [Figure 4]FIG. 4 is a graph showing the effective productivity of cultures BBi Wildtype C1 stored for X=7 d (dotted grey) or X=14 d (solid grey) and BBi Heat 6.6 stored for X=7 d (dark upward diagonal stripes) or X=14 d (light upward diagonal stripes) at 50° C. and low relative humidity. [Diagram 5] FIG. 5 is a graph showing cell dry weight concentrations measured at harvest in cultures of BBi Wildtype C1 (solid dark grey), BBi KCl 1.2 (downward diagonal stripes), BBi KCl 5.5 (upward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots). [Figure 6] FIG. 6 is a graph showing the percent cell viability after downstream processing of cells from cultures BBi Wildtype C1 (solid dark grey), BBi KCl 1.2 (downward diagonal stripes), BBi KCl 5.5 (upward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) compared to the fermentation cell number after conditioning (CN). [Figure 7] FIG. 7 is a graph showing the viability, as a percentage of cells before storage (“T0”), of BBi Wildtype C1 (solid dark grey), BBi KCl 1.2 (downward diagonal stripes), BBi KCl 5.5 (upward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) cells stored at 30° C. and 40% relative humidity for X=14 d (light borders) or X=28 d (dark borders). [Figure 8]FIG. 8 is a graph showing the effective productivity of cultures BBi Wildtype C1 (solid dark grey), BBi KCl 1.2 (downward diagonal stripes), BBi KCl 5.5 (upward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) stored at 30° C., 40% relative humidity for X=14 d (light borders) or X=28 d (dark borders). [Figure 9] FIG. 9 is a graph showing the time course of cumulative base addition for BBi Wildtype C1 (solid line), BBi Heat 4.1 (short dash line), and BBi Heat 6.6 (long dash line) cultures. [Figure 10] FIG. 10 is a graph showing the effective productivity of cultures BBi Wildtype C1 (solid grey), BBi Heat 4.1 (downward diagonal stripes), and BBi Heat 6.6 (upward diagonal stripes) stored at 30° C. and 40% relative humidity for 14 d. [Figure 11] FIG. 11 is a graph showing the effective productivity of cultures BBi Wildtype C1 (solid grey), BBi Heat 4.1 (downward diagonal stripes), and BBi Heat 6.6 (upward diagonal stripes) stored at 30° C. and low relative humidity for 137 d. [Figure 12] FIG. 12 is a graph showing the time course of cumulative base addition for BBi Wildtype C1 (solid line), BBi KCl 1.2 (dashed line), BBi NaCl 2.7 (short dash line), BBi NaCl 3.3 (long dash line), BBi NaCl 5.1 (long dash line followed by two dots), and BBi NaCl 6.2 (long dash line followed by one dot). [Figure 13]FIG. 13 is a graph showing cell dry weight concentrations measured at harvest in cultures of BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots). [Figure 14] FIG. 14 is a graph of cell dry weight concentration measured at harvest in cultures of BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots). [Figure 15] FIG. 15 is a graph showing the percent cell viability following downstream processing of cells from cultures BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) compared to the fermentation cell number after conditioning (CN). [Figure 16] FIG. 16 is a graph of the percent of cells prior to storage ("T0") of cell viability for cultures BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) stored at 30° C. and 40% relative humidity for 14 d. [Figure 17]FIG. 17 is a graph showing cell viability, as a percentage of cells before storage ("T0"), of cultures BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) stored at 30° C. and low relative humidity for 137 d. [Figure 18] FIG. 18 is a graph showing cell viability, as a percentage of cells before storage ("T0"), of cultures BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) stored at 50° C. and low relative humidity for 14 d. [Figure 19] FIG. 19 is a graph showing the effective productivity of cultures BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) stored at 30° C. and 40% relative humidity for 14 d. [Figure 20] FIG. 20 is a graph showing the effective productivity of cultures BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) stored at 30° C. and low relative humidity for 137 d. [Figure 21]FIG. 21 is a graph showing the effective productivity of cultures BBi Wildtype C1 (solid grey), BBi KCl 1.2 (downward diagonal stripes), BBi NaCl 2.7 (50% dots), BBi NaCl 3.3 (60% dots), BBi NaCl 5.1 (70% dots), and BBi NaCl 6.2 (80% dots) stored at 50° C. and low relative humidity for 14 d. [Figure 22] FIG. 22 is a graph showing the time course of cumulative base addition for NCFM Wildtype C1 (solid line), NCFM Wildtype C2 (dashed line), NCFM Heat 1.1 (long dash line), and NCFM Heat 6.3 (long dash line) cultures. [Figure 23] FIG. 23 is a graph showing cell dry weight concentrations measured at harvest in cultures of NCFM Wildtype C1 (solid grey), NCFM Wildtype C2 (light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes). [Figure 24] FIG. 24 is a graph showing cell concentration at harvest and subsequent storage at 10C for 1 h in cultures of NCFM Wildtype C1 (solid grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes). [Diagram 25] FIG. 25 is a graph showing the percent cell viability following downstream processing of cells from cultures NCFM Wildtype C1 (solid grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) compared to the fermentation cell number after conditioning (CN). [Figure 26]FIG. 26 is a graph showing cell viability as a percent of cells before storage ("T0") for cultures NCFM Wildtype C1 (solid grey), NCFM Wildtype C2 (light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) after 14 d (light borders) and 28 d (dark borders) of storage at 30° C. and 40% relative humidity. [Figure 27] FIG. 27 is a graph showing cell viability, as a percentage of cells before storage ("T0"), of cultures NCFM Wildtype C1 (solid dark grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) after 7 d (light borders) and 14 d (dark borders) of storage at 50° C. and low relative humidity. [Figure 28] FIG. 28 is a graph showing the effective productivity of cultures NCFM Wildtype C1 (solid dark grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) stored at 30° C., 40% relative humidity for X=14 d (light borders) or X=28 d (dark borders). [Figure 29] FIG. 29 is a graph showing the effective productivity of cultures NCFM Wildtype C1 (solid dark grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) stored at 50° C. and low relative humidity for X=7 d (light borders) or X=14 d (dark borders). [Diagram 30] FIG. 30 is a graph showing the time course of cumulative base addition for NCFM Wildtype C1 (solid line), NCFM Wildtype C2 (dashed line), NCFM Heat 1.1 (long dash line), and NCFM Heat 6.3 (long dash line) cultures. [Diagram 31]FIG. 31 is a graph showing cell dry weight concentrations measured at harvest in cultures of NCFM Wildtype C1 (solid grey), NCFM Wildtype C2 (light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes). [Diagram 32] FIG. 32 is a graph showing cell concentration at harvest and subsequent storage at 10C for 1 h in cultures of NCFM Wildtype C1 (solid grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes). [Diagram 33] FIG. 33 is a graph showing percent cell viability following downstream processing of cells from cultures NCFM Wildtype C1 (solid grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) compared to fermentation cell number after conditioning (CN). [Diagram 34] FIG. 34 is a graph showing cell viability, as a percentage of cells before storage ("T0"), of cultures NCFM Wildtype C1 (solid grey), NCFM Wildtype C2 (light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) stored at 30° C. and 40% relative humidity for 28 d. [Diagram 35] FIG. 35 is a graph showing cell viability, as a percentage of cells before storage ("T0"), of cultures NCFM Wildtype C1 (solid dark grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) stored at 30° C. and low relative humidity for 4 m. [Diagram 36]FIG. 36 is a graph showing cell viability, as a percentage of cells before storage ("T0"), of cultures NCFM Wildtype C1 (solid dark grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) stored at 50° C. and low relative humidity for 14 d. [Figure 37] FIG. 37 is a graph showing the effective productivity of cultures NCFM Wildtype C1 (solid dark grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) stored at 30° C. and 40% relative humidity for 28 d. [Figure 38] FIG. 38 is a graph showing the effective productivity of cultures NCFM Wildtype C1 (solid dark grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) stored at 30° C. and low relative humidity for 4 m. [Figure 39] FIG. 39 is a graph showing the effective productivity of cultures NCFM Wildtype C1 (solid dark grey), NCFM Wildtype C2 (solid light grey), NCFM Heat 1.1 (vertical stripes), and NCFM Heat 6.3 (horizontal stripes) stored at 50° C. and low relative humidity for 14 d. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The following sequences comply with 37 CFR § 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures-the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998), EPO and PCT sequence listing requirements (Rules 5.2 and 49.5(a-bis)), and Part 208 and Annex C of the Administrative Instructions. The symbols and format used for the nucleotide and amino acid sequence data comply with the rules set forth in 37 CFR § 1.822.

[0029] SEQ ID NO:1 is the nucleotide sequence of the genome of the ancestral strain, Bifidobacterium animalis lactis, Bi-07, corresponding to Genbank accession number NC_017867. SEQ ID NO:2 is the nucleotide sequence of the genome of the ancestral strain, Lactobacillus acidophilus NCFM®, corresponding to Genbank accession number NC_006814. SEQ ID NO:3 is the first portion of the genomic sequence of the ancestral strain of Lactobacillus paracasei, Contig A. SEQ ID NO:4 is the second part of the genomic sequence of the ancestral strain of Lactobacillus paracasei, Contig B. SEQ ID NO:5 is the sequence of a plasmid present in the genome of an ancestral strain of Lactobacillus paracasei.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (by Singleton and Sainsbury) (Singleton and Sainsbury, 2006) and THE HARPER COLLINS DICTIONARY OF BIOLOGY (by Hale and Marham) (Hale and Marham, 1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0031] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges are inclusive of the numbers defining the range.

[0032] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which aspects or embodiments can be understood by reference to the specification as a whole, and accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0033] Other definitions of terms may appear throughout this specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0034] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value between the upper and lower limit of that range is also understood to be specifically disclosed to the first decimal place of the unit of the lower limit. Each narrower range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these narrower ranges may independently be included or excluded in the range, and each range where either or both limits are included in the narrower range, or where both limits are not included, is also encompassed within the disclosure, provided that any limit is specifically excluded from the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0035] For the interpretation of the claims and this specification, the following definitions and abbreviations should be used.

[0036] The term "invention" or "present invention" as used herein is an open-ended term and is not intended to refer to any single embodiment of a particular invention, but rather encompasses all possible embodiments described in the specification and claims.

[0037] The term "about" used herein to modify the amount of an invention component or reactant used refers to variations in the numerically indicated amount that may occur, for example, due to typical measuring procedures and liquid handling procedures used to make concentrates or use solutions in the real world; due to inadvertent errors in these procedures; due to differences in the manufacture, source, or purity of the components used to make the composition or to carry out the method. The term "about" also encompasses amounts that differ due to various equilibrium conditions for the composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents of the amount. In one embodiment, the term "about" means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

[0038] The abbreviation "ATCC" refers to the American Type Culture Collection International Depository, Manassas, VA, USA. "ATCC Number" refers to the accession number of the culture deposited with ATCC.

[0039] The abbreviation "DSM" refers to the accession number of the culture deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ).

[0040] As used herein, the terms "comprises," "including," "includes," "including," "having," "having," "containing," or "containing" or any other variations thereof are intended to cover non-exclusive inclusions. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or device. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, condition A or condition B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and A and B are both true (or exist). Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be open ended with regard to the number of instances (i.e., occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and singular forms of elements or components include plurals unless the number is clearly intended to be singular.

[0041] The terms "deletion" or "disruption" when used in reference to genes, genetic constructs, etc., refer to the partial or complete inactivation of a normally functioning nucleic acid sequence. A deletion in a sequence refers to the removal of all or part of the sequence, which may result in a complete or partial inactivation of the sequence. A disruption or insertion in a sequence refers to the addition of an element within the sequence, which again will reduce or eliminate the ability of the sequence to function normally. A deletion or disruption will render the gene or coding sequence "non-functional" within the meaning of the present invention.

[0042] "Gene" refers to a nucleic acid fragment that expresses a specific protein, and may refer to the coding region alone or may include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0043] "Intergenic region" means a stretch of DNA sequence located between genes. An intergenic region is a subset of non-coding DNA.

[0044] "Genome" refers to the collection of polynucleotide sequences that contain all the genetic information that defines an organism. The genomic sequence of an organism includes both genes and non-coding sequences of DNA. Typically, genome as used herein will refer to a prokaryotic genome contained on a single chromosome.

[0045] "Phenotype" refers to a detectable characteristic of a cell or organism. "Beneficial phenotype" refers to an evolved cellular phenotype that enhances its storage stability, effective production capacity (including enhanced cell proliferation, resistance to stressors or stress conditions, including but not limited to, resistance to heat, resistance to osmotic pressure, resistance to surfactants, and resistance to oxidative stress).

[0046] "Polynucleotide", "nucleic acid sequence", "nucleotide sequence", or "nucleic acid fragment" are used interchangeably to refer to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) that is single- or double-stranded, optionally containing synthetic, non-natural, or modified nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by the single letter designations: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide. "Regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that affects the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include, but are not limited to, promoters, translation leader sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0047] The term "bacteria" or "bacterial" or "prokaryotic" is used herein to refer to any bacterial species, strain, or combination thereof, and is not limited to strains currently recognized as probiotics. However, the bacterial strains used in the present invention are suitable for human and / or animal consumption. Those skilled in the art will be readily aware of specific species and / or strains within the genera described herein that are used in the food and / or agricultural industries and are generally considered suitable for human and / or animal consumption. Such bacterial strains are typically non-pathogenic and may generally be considered safe for human use (e.g., GRAS). The term "bacteria" is generally used to refer to all bacteria, e.g., all viable bacteria. Bacteria suitable for use in the present invention include, but are not limited to, the genera Bifidobacterium, Lactobacillus, Brevibacterium, Propionibacterium, Lactococcus, and Streptococcus.

[0048] As used herein, the term "mutant" refers to any microorganism resulting from the modification of a parent (i.e., deposited) strain. For example, a mutant can be a microorganism resulting from a genetic modification of the deposited strain.

[0049] The term "mutation" refers to a change in the nucleotide sequence of an organism and may include the substitution of one or more nucleotides with one or more different nucleotides, or the deletion or insertion of one or more nucleotides. Within the context of the present invention, mutations may occur naturally or as a result of the presence of stress conditions.

[0050] The term "Bifidobacterium" refers to gram-positive, non-acid-fast bacteria. The bacteria are pleomorphic, cornyform, rod-shaped bacteria that usually appear clubbed or branched. The bacteria are usually non-motile and non-spore forming. Species of the genus Bifidobacterium include B. actinocoloniiforme, B. adolescentis, B. angulatum, B. animalis, B. aquikefiri, B. asteroids, B. biavatii, B. B. bifidum, B. bohemicum, B. bombi, B. boum, B. breve, B. callitrichos, B. catenulatum, B. choerinum, B. commune, B. coryneforme, B. cuniculi, B.crudilactis, B.denticolens, B.dentium, B.eulemuris, B.faecale, B.gallicum, B.gallinarum, B.hapali, B.indicum, B.inopinatum, B. B. kashiwanohense, B. infantis, B. lemurum, B. longum, B. magnum, B. merycicum, B. minimum, B. mongoliense, B. moukalabense, B. myosotis, B.Examples of the species include B. pseudocatenulatum, B. pseudolongum, B. psychraerophilum, B. pullorum, B. reuteri, B. ruminantium, B. saguini, B. scardovii, B. stellenboschense, B. stercoris, B. saeculare, B. subtile, B. thermocidophilum, B. thermophilum, B. tissieri, and B. tsurumiense. A preferred subspecies of the present invention is B. animalis subsp. lactis. A preferred Bifidobacterium strain of the present invention is B. animalis subsp. lactis Bi-07. As used herein, "Bifidobacterium Bi-07, ATCC PTA SD5220" refers to the ancestral Bifidobacterium strain from which the BBi strains of the present invention evolved.

[0051] The term "Lactobacillus" refers to a genus of rod-shaped, non-spore-forming, Gram-positive, facultative anaerobic bacteria with a DNA base composition of less than 53% G+C. The genus is usually non-respiratory and lacks catalase. The genus ferments glucose primarily to lactic acid, or to lactic acid, CO2, and ethanol. Normally, Lactobacillus grows anaerobically, but unlike most anaerobes, it grows as an "aerotolerant anaerobe" in the presence of O2. The genus lacks catalase, but does have superoxide dismutase and has an alternative means of detoxifying peroxide radicals, usually by the enzyme peroxidase. Species of the genus Lactobacillus include L. acetotolerans, L. acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L. alimentarius, L. amylolyticus, L. amylophilus, L. amylotrophicus, L. amylovorus, L. animalis, L. antri, L. apodem. (L.apodeme), L.aviaries, L.bifermentans, L.brevis, L.buchneri, L.camelliae, L.casei, L.catenaformis, L.ceti, L.coleohominis, L.collinoides, L.composti, L.concavus, L.coryniformis, L.crispatus, L.crustorum, L.curvatuscurvatus, L. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. dextrinicus, L. diolivorans, L. equi, L. equigenerosi L. equigenerosi, L. farraginis, L. farciminis, L. fermentum, L. fornicalis, L. fructivorans, L. frumenti, L. fuchuensis, L. gallinarum, L. gasseri, L. gastricus, L. gannensi L.ghanensis, L.graminis, L.hammesii, L.hamster, L.harbinensis, L.hayakitensis, L.helveticus, L.hilgardii, L.homohiochii, L.iners, L.ingluviei, L.intestinalis ntestinalis, L. jensenii, L. johnsonii, L. kalixensis, L. kefiranofaciens, L. kefiri, L. kimchii, L. kitasatonis, L. kunkeei, L. leichmannii, L. lindneri, L. malefermentans, L.malefermentans)、L.マリ(L.mali)、L.マニホチボランス(L.manihotivorans) (L. mindensis)、L. mucosae (L. murinus)、L. .nagelii)、L.ナナレレシス(L.namurensis)、L.nantensis)、L.オリゴフェ(L.oligofermentans)、L.oligofermentans (L.oris)、L.panis)、L. L.pantheris)、L.parabrevis)、L.parabrevis (L.parabuchneri)、L .パラカゼイ(L.paracasei)、L.パラコリノイデス(L.paracollinoides)、L. parapharraginis) (L.paralimen tarius)、L.paraplantarum (L. pentosus)、L. pentosus (L.perolens)、L.plantarum (L.pontis)、L. (L.protectus)、L.psittaci (L.psittaci)、L.rennini)、L.re uteri)、L.ラムノサス(L.rhamnosus)、L.リマエ(L.rimae)、L.ロゴサエ(L.rogosae)、L .ロシアエ(L.rossiae)、L. (L.sakei)、L.salivarius (L.salivarius)(L.sanfranciscens is) L. satsumensis (L. secaliphilus)、L. L.sharpeae (L.siliginis)、L.spicheri)、L.s uebicus)、L.タインデンシス(L.thailandensis)、L.ウルツネンシス(L.ultunensis)、L.Examples of Lactobacillus include L. vaccinostercus, L. vaginalis, L. versmoldensis, L. vini, L. vitulinus, L. zeae, and L. zymae. As used herein, "Lactobacillus acidophilus NCFM ATCC 700396" refers to the ancestral strain of Lactobacillus acidophilus NCFM from which the Lactobacillus acidophilus strains of the present invention evolved.

[0052] The term "adaptation" describes the process by which organisms become better suited to their environment through the inheritance of beneficial traits. Adaptation is a multi-generational process in which beneficial mutations arise in a population and can increase in frequency over time due to the benefit they confer. In sexually reproducing organisms, beneficial traits from different parents can be recombined through sexual recombination and carried forward together into progeny.

[0053] The term "selection" describes the process that results in changes in allele frequencies within a population over time as a result of competition: beneficial alleles increase the competitive fitness, leading to increased reproduction and survival of individuals carrying those alleles, resulting in an increase in the frequency of those beneficial alleles in a population over time.

[0054] The term "genetic variation" describes the total set of genetic mutations in all genes within an assembly of distinct individuals or within a population. Clonal populations lack genetic variation. Genetic variation within a population can be increased by processes such as mutation, recombination, hybridization, horizontal gene transfer, and copy number variation, to name a few. Genetic variation can be decreased by selection or genetic drift (due to random sampling error).

[0055] The term "experimental evolution" describes a method to replicate the process of natural selection in the lab. Most experiments performed rely on a combination of selection by competition and the generation of genetic variation by natural mutation rates, resulting in individuals with increased fitness in a selective environment, better adapted than their ancestors. The process of natural selection is repeated by working with large population sizes and over many generations (100+). Experimental evolution may also include experiments in which the fitness of the evolving population decreases, which is often the case in mutation accumulation experiments where the population is bottlenecked (reduced to single cells or sibling mate pairs) to reduce competition leading to the accumulation of a random set of mutations (not beneficial mutations). Experimental evolution experiments can be performed in a variety of selection regimes, including but not limited to batch cultures (flask transfer), continuous culture vessels (e.g., chemostat, turbidostat, morbidostat), agar plates, or microfluidic droplets.

[0056] The term "ancestor" or "ancestor strain" describes an individual from which individuals with similar but different genotypes are derived. This may be the common ancestor of two phylogenetically related species, or the starting clone of an experimental evolution experiment. Here, the term "ancestor" is used to refer to the original clone used to seed the replicate population of an evolution experiment. In this case, the "ancestor" is the clone currently sold as a probiotic as part of the HOWARU® brand.

[0057] The term "evolutionary time" refers to the time frame over which evolutionary processes, such as adaptation, occur, which may require hundreds or thousands of generations. "Evolutionary time" differs from ecological time, which is much shorter, because ecological processes occur much faster than evolutionary processes.

[0058] The term "spontaneous mutation" refers to a mutation that occurs due to an error during replication, DNA damage, or DNA repair. Spontaneous mutations depend on the spontaneous mutation rate, which describes how often a nucleotide changes in a genome per generation and genome, or how often deletions or insertions occur in a genome per generation and genome. Spontaneous mutation rates are adaptive traits and vary between organisms and environments.

[0059] The term "stressor" refers to any environmental stress to which a bacterial strain can be exposed and which, over time, will result in genetic changes in the bacteria. Stressors of particular interest in the present invention are those associated with production protocols for probiotics and include, but are not limited to, salt or osmotic stressors, heat stressors, oxidative stressors, and acid stressors. Strains exposed to heat stress are referred to herein as "thermo-evolved." Strains exposed to osmotic stress are referred to herein as "salt-evolved."

[0060] The term "storage stability" characterizes the ability of a cell population to maintain viability during storage. Cells in this context are considered viable if they are able to grow and proliferate again once transferred to permissive conditions. Assay methods to evaluate storage stability include, but are not limited to, Most Probable Number (MPN) analysis, or plating and counting colony forming units (CFU). Due to the complexity of cells, the loss of viability of a cell population can be described as a stochastic process at the level of a single cell. However, for a cell population, the expected decay rate or fractional decay of the viable cell population can be characterized (e.g., by determining the MPN or CFU concentration at the beginning and end of a storage experiment) and is often used interchangeably with the term storage stability. In cases where a cell population consists primarily of monoclonal cells, the determined decay rate of the MPN or CFU count, or the drop in the fraction of viable cells, is used interchangeably with the storage stability of the strain.

[0061] The storage stability of cells can be tested under a variety of conditions, with or without formulation, and with or without specific packaging. Important determinants of conditions include, but are not limited to, temperature, relative humidity, chemical composition (e.g., oxygen content in the gaseous atmosphere), and radiation levels of the environment. Storage stability also depends on the properties of the cells (e.g., the type of strain used), the way the cells are produced, and the downstream unit operations in which the cells are processed.

[0062] Conditions for testing the storage stability of beneficial microorganisms, even if not intended for use as active pharmaceutical ingredients, usually follow the guidance set out in the document "Stability testing of active pharmaceutical ingredients and finished pharmaceutical products" published as Annex 2 of the World Health Organization (WHO) Technical Report Series, No. 953, 2009(1) and the ICH guideline "Q1A(R2): Stability Testing of New Drug Substances and Products" published by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) in 2003, both of which are incorporated herein by reference.

[0063] To standardize testing for storage stability, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) has divided the world into five different zones. Zone I Temperate Zone II Mediterranean / Subtropical Zone III Hot and dry zone Zone IVa Hot and humid / Tropical Zone IVb Hotter / More Humid

[0064] The climatic conditions for these zones in long term storage stability testing are usually reflected as follows: Zone I 21℃±2℃ 45%RH±5%RH Zone II 25℃±2℃ 60%RH±5%RH Zone III 30℃±2℃ 35%RH±5%RH Zone IVa 30℃±2℃ 65%RH±5%RH Zone IVb 30℃±2℃ 75%RH±5%RH

[0065] Also, to "speed up" the decay process and allow for faster testing of storage stability within shorter time periods, frequently temperatures higher than the 30°C ± 2°C set forth in the ICH long-term study recommendations mentioned above are used to assess storage stability, including, but not limited to, 38°C ± 2°C, 40°C ± 2°C, or 50°C ± 2°C.

[0066] The term "critical process parameter" or "CPP" refers to a key variable that is set and / or controlled to affect the manufacturing process of a live cell product. Key CPPs in a fermentation process include, but are not limited to, (i) pH, (ii) temperature, (iii) stirrer speed, (iv) gas input, (v) elapsed process time, (vi) inoculum ratio, (vii) medium composition, (viii) medium formulation, and (ix) composition of gas input.

[0067] The term "effective productivity" defines a criterion for productivity measurement. "Effective productivity" relates the classical concept of "productivity" that characterizes the number of cells produced per volume and time in a production process to the stability of the produced cells as a formulated product on shelf at specified conditions. The total time (t total ) is the time for preparing the fermentation mass (t prep ), (ii) the time from the inoculum of the fermentate to the harvest of the fermentate (t grow), and (iii) the time required for downstream processing of the cells and formulation into a final product (t form ) and can be determined according to: t total =t prep +t grow +t form

[0068] Total cell product (Δcp total ) is the number of viable cells at the end of the storage period from a given volume of last stage fermenter, corrected for the fraction of cell product (seed vial and / or inoculum) used to start the process. From these key metrics, the "effective productivity" (p effective ) can be derived according to:

number

[0069] Typically, the effective productivity refers only to the last stage fermenter. Also, frequently, downstream unit operations of the process and formulation unit operations can be ignored since they are kept the same and / or exhibit fairly similar time requirements. Last but not least, the amount of inoculum introduced into the process is usually insignificant when compared to the final cell concentration in the fermenter at harvest and can therefore be ignored in a first approximation, simplifying the formula to:

number

[0070] The term "probiotic" as used herein refers to live microorganisms that, when administered in appropriate amounts, confer a health benefit to the recipient. Probiotics are suitable for human consumption and are non-pathogenic and non-toxic. Probiotic strains generally have the ability to survive the passage through the upper part of the digestive tract. Probiotic bacteria typically exert beneficial effects on health, on the one hand, through their ecological interaction with the resident flora in the digestive tract, and on the other hand, through their ability to positively influence the immune system via the "GALT" (gut-associated lymphoid tissue). Probiotic bacteria, if given in sufficient numbers, have the ability to survive and progress through the intestine. However, since probiotic bacteria do not cross the intestinal barrier, their main effect is induced within the lumen and / or wall of the digestive tract. Probiotic bacteria then form part of the resident flora during the administration period. This colonization (or transient colonization) allows probiotic bacteria to exert beneficial effects such as suppression of potentially pathogenic microorganisms present in the microbiota and interaction with the immune system of the intestine.

[0071] The present invention relates to novel evolved strains of the genera Bifidobacterium and Lactobacillus with unique genetic profiles that demonstrate enhanced storage stability or increased effective productivity. Storage stability was measured by a series of unique assays. This allowed the applicant to select evolved strains with surprising increases in storage stability and effective productivity. The present invention additionally provides a method for the experimental evolution of probiotic strains to develop underlying genetic modifications that are specifically linked to increased storage stability.

[0072] Probiotic Strains: Genus Bifidobacterium: The present invention provides evolved strains of the genus Bifidobacterium with improved storage stability. Bifidobacteria constitute the predominant gut microbiota in infants and are also abundant in the adult population, constituting up to 10% of the normal gut microbiota, but numbers begin to decline in the elderly. Individuals are typically colonized with one to four Bifidobacterial species (Maettoe et al., 2004). In addition to individual variation, the composition of Bifidobacterial species varies between different age groups. B. longum biovars infantis, breve, and bifidum are the most prevalent species in infants, and B. longum biovars longum, adolescentis, bifidum, and catenulatum in adults. Variations in the number (Mueller et al., 2006) and species composition (Maettoe et al., 2004) of bifidobacteria between geographic regions have also been reported. Bifidobacteria are usually considered health-promoting bacteria, and increasing bifidobacterial numbers in the intestinal tract is typically used as an endpoint in gut health targeting products such as probiotics and prebiotic intervention studies.

[0073] Strains of the Bifidobacterium genus species are used as probiotics. However, due to technical challenges related to the stability of the genus, only a few prominent species and strains, mainly B. animalis subspecies lactis, are currently available on the market. Bifidobacteria or Bifidobacteria-containing strain mixtures have shown promising results, for example, in alleviating symptoms of irritable bowel syndrome (Brenner and Chey, 2009), inflammatory bowel disease (Macfarlane et al., 2009), diarrhea (Chouraqui et al., 2004), eczema (Kim et al., 2010), asthma (Yoo et al., 2007), and colds (de Vrese et al., 2005).

[0074] Therefore, Bifidobacterium spp. are prime targets for probiotic applications, and strains with increased storage stability would be of particular interest. Any species of the genus Bifidobacterium can be used in the present invention and subjected to the experimental evolution protocol described herein, with the species Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium bifidum, and Bifidobacterium adolescentis being preferred, with Bifidobacterium animalis being preferred. Most preferred are strains of Lactis animalis, particularly strain Bi-07. Bi-07 is commercially available from DuPont and has been deposited with the ATCC under accession number PTA-SD5220. Bi-07 has been fully sequenced and the sequence is available under accession number NC_017867.

[0075] Lactobacillus genus In addition to evolved strains of the Bifidobacterium genus, the present invention provides strains of the Lactobacillus genus that demonstrate increased storage stability. Any species of the genus Lactobacillus may be used in the present invention and subjected to the experimental evolution protocol described herein, with the species L. acidophilus, L. curvatus, L. rhamnosus, L. casei, L. paracasei, L. salivarius, L. reuteri, L. salivarius, L. gallinarum, L. crispatus, L. agilis being preferred, and the species Lactobacillus paracasei and Lactobacillus acidophilus NCFM being most preferred.

[0076] Lactobacillus paracasei is a gram-positive, non-spore-forming rod-shaped bacterium that is a common inhabitant of the human intestinal tract. L. paracasei strains are also found naturally in fermented vegetables, milk, and meat. Strains of this species are used in many foods, including traditional fermented milk and cheese. Selected strains of this species are also used in probiotic foods and dietary supplements. Lactobacillus paracasei Lpc-37 is commercially available from DuPont. It has been genetically characterized and properly classified as Lactobacillus paracasei by 16S rRNA gene sequencing. L. paracasei Lpc-37 is a strain isolated from a dairy source and has been deposited at the American Type Culture Collection as SD5275. Its genome has been fully sequenced and is available at NCBI under the accession number NOKL00000000. Lactobacillus acidophilus is a species of Gram-positive bacteria within the genus Lactobacillus. L. acidophilus is a homofermentative, microaerophilic species that ferments sugars to lactic acid and grows readily at fairly low pH values ​​(below pH 5.0), with an optimal growth temperature of approximately 37°C (99°F). L. acidophilus occurs naturally in the gastrointestinal tract and oral cavity of humans and animals and has been shown to have probiotic efficacy. Its genome has been sequenced. Lactobacillus acidophilus NCFM® was isolated from a human fecal sample in the early 1970s. The strain designation NCFM® comes from "North Carolina Food Microbiology," the research laboratory at North Carolina State University (NCSU) where the isolation took place.The strain has been shown to have beneficial effects in humans when taken orally and has been sold commercially for over 25 years. The strain is available from ATCC as 700396. The genome has been fully sequenced and is available from NCBI under accession number NC_006814.

[0077] Experimental evolution protocol The method of the present invention utilizes an experimental evolution protocol to evolve various probiotic strains that achieve enhanced or improved storage stability. The protocol involves repeatedly subjecting ancestral strains to various stresses to generate genetic changes in the strains that are phenotypically manifested as increased resistance to the stress and increased storage stability of the strain. Various stressors may be used in the experimental evolution protocol, including heat, salt, detergent, and acid or base. A typical experimental evolution protocol begins with the selection of a specific ancestral strain with well-documented probiotic properties. Each strain is grown in an appropriate medium, plated on agar plates, and single colonies are isolated, grown, and frozen to generate a bank of frozen stocks. The single colony from the stock is then used to find the repeat population, which is then cultured and exposed to various concentrations of stressor for multiple generations, expected to generate an evolved strain for at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 generations. Those skilled in the art will recognize that depending on the stressor used and the underlying genetic characteristics of the strain, culturing beyond 100 or 300 or 400 or 500 generations may be required to evolve the strain. The concentration of the stressor may be gradually increased during the selection experiment. In some situations, the mutation rate can be increased by exposing the mutagen to the stressor in alternating combinations. Such mutagenists are common and well known in the art and include, but are not limited to, UV exposure, chemical mutagenesis (such as, but not limited to, ethyl methanesulfonate, nitrous acid, ethidium bromide, mitomycin C), and transposon-mediated mutagenesis.

[0078] When heat is used as the stressor, the exposure temperature should be above the standard growth temperature of the wild-type or ancestral strain, preferably 1°C to 20°C above the standard growth temperature. The optimal temperature for selecting evolved strains will vary depending on the bacteria selected and the progress of the selection experiment. For example, if the strain is a species of the genus Bifidobacterium, the selection temperature may be about 41°C to about 50°C.

[0079] Similarly, when osmotic stress is introduced by exposure to salt, the concentration of the salt will vary depending on the compound used and the growth characteristics of the bacteria. Various salts may be used as stressors in the present invention, including but not limited to NaCl, KCl, CaCl2. For example, when NaCl or KCl is used, the concentration may range from at least about 0.2% to about 1.2%, or at least about 0.4% to at least about 1.0%, or at least about 0.5% or at least about 0.5% to at least about 0.8% in the growth medium, with at least about 0.4% to at least about 0.8% being preferred.

[0080] Surface active agents such as detergents may be used to introduce cell envelope stress into the bacterial population. Surface active agents are well known and commonly used, including but not limited to anionic detergents such as sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), ammonium lauryl sulfate (ALS), or their ethoxylated companions, sodium laureth sulfate (SLES); or cationic detergents such as amines, alkyl imidazolines, alkoxylated amines, and quaternized ammonium compounds; or amphoteric detergents, or non-ionic detergents. Preferred for use in the present invention is SDS, and typical concentrations of the detergent will be close to sublethal concentrations, ranging from about 0.008% to about 0.01% in the growth medium.

[0081] Cells undergoing experimental evolution are grown in liquid medium and diluted to a specific OD in fresh culture broth supplemented with stressors, if applicable, or exposed to higher temperatures. Typically, clonal populations are transferred every 24-48 hours, after which the average OD of the population is determined. The number of generations per day is calculated based on the following formula:

number

[0082] The number of generations is monitored. If an increase in population density is observed compared to the ancestral strain, the selection pressure is increased. If the number of generations is observed to decrease and the population is unable to make up for dilution upon transfer, the selection pressure may be reduced to maintain a viable population. In this way, the selection pressure is maintained at the highest possible concentration of stressor over the life of the evolutionary process. After completion of the evolutionary process, the population may be frozen for further study.

[0083] Assay to identify improved strains Several assays are used in the present invention to determine the relative storage stability or effective production capacity of the evolved strains.

[0084] Spot plating assay A spot plating assay was used as a first screen to identify stress evolved clones based on improved storage stability. To identify improved strains, single colonies were isolated from the evolved population and tested for improved viability during storage under stress conditions. Stress conditions spanned a wide range of temperatures (typically 4C-50C) and a wide range of different levels of humidity (0-65%) in the spot plating assay (see General Methods for a detailed description of the spot plating assay). Isolated clones were grown as single clones. A standard amount of cells was mixed with stabilizer buffer. Stable cells were flash frozen and lyophilized before exposure to storage conditions. At regular intervals, the stored cell pellets were rehydrated. Selection was performed using the same medium but without any stressor, and serial 10-fold dilutions were plated on agar plates. Viability could also be assessed using a most probable number assay (MPN). The highest dilution at which the stored sample was still able to grow was recorded. The most viable clones were selected from the spot plating assay and subjected to an acceleration assay to obtain a more accurate measure of the clones' ability to survive various long-term storage conditions. Clones tested in the acceleration assay were sequenced using whole genome sequencing to identify mutations that had occurred during the selection experiment.

[0085] Promotion assay The clones identified in the previous spot plating assay were subjected to sequential assays to evaluate storage stability and effective productivity. Briefly, the evolved clones and their corresponding ancestral strains (as controls) were grown in bioreactors under controlled conditions of pH, temperature, and stirrer speed. After bioreactor cultivation, the clones were subjected to small-scale plate assays to determine storage stability and effective productivity under various storage conditions, including storage in "wet" conditions at 25°C, 30°C, or 40°C, and with relative humidity that may range from about 35% to about 40% to about 60% to about 70% or to about 75%, or alternatively storage under "dry" conditions, where the cells are stored at about 50°C and less than about 1% relative humidity. The storage stability and effective productivity of the evolved clones relative to the ancestral strains were determined based on dry cell weight and CFU determinations.

[0086] Preferred Embodiments An evolved strain of Bifidobacterium animalis subspecies lactis Bi-07 (BBi) The BBi strain evolved in the presence of several stressors, including high temperature, salts (KCl, NaCl), detergents (SDS), and oxidants (peroxides). Although exposure to peroxides and SDS showed inconclusive results, clones evolved against osmotic and thermal stresses demonstrated clear improvements in storage stability and effective production capacity compared to the ancestral strain.

[0087] Thermally evolved clones At least three thermally evolved populations had clones with improved storage stability. Two clones were selected from two different populations (BBi Heat 4.1 and BBi Heat 6.6) and their storage stability was tested under wet and dry conditions in an accelerated assay. The CFU at the end of fermentation (end of run, EOR) of the evolved strains was not different from the unevolved control, but the evolved clones took about 2 hours longer to reach the end of fermentation than the unevolved control. One clone, Heat 4.1, showed a >100% improvement in viability in wet conditions and a 68% improvement in dry conditions. The other clone, Heat 6.6, also showed improvement. Considering the overall viability (including viability during downstream processing and storage), both Heat 4.1 and Heat 6.6 performed fairly well in wet conditions for 14 days. After 137 days in dry conditions at 30°C, the number of colony forming cells of the evolved strains is 5-50x better than the ancestral strain. In dry conditions, only Heat 4.1 shows an improvement in overall survival after 14 days.

[0088] Salt evolved clones Seven clones from six NaCl and six KCl evolved populations were tested for improved storage stability using spot plating assays. Overall, all six KCl evolved populations had at least one clone with increased storage stability, while five NaCl evolved populations had more stable clones (see Examples). Of these more stable clones, two KCl evolved, KCl 1.2 and KCl 5.5, and four NaCl evolved strains, NaCl 2.7, NaCl 3.3, NaCl 5.1, and NaCl 6.2, were tested in accelerated assays. All clones had higher CFU counts at the end of fermentation relative to the unevolved control. Considering all fermentations, none of the salts evolved clones with improved storage stability over 14 days in dry conditions. However, under moist storage conditions, an overall improvement in viability was observed in all but one salt evolved clone after 14 days.

[0089] Physiological differences of evolved clones at the end of fermentation The evolved clones were analyzed for changes in energy content, as well as organic and fatty acid composition, relative to the ancestral strain. The energy content of the cells was significantly different at the end of fermentation. Interestingly, both storage-stable thermally evolved clones had higher energy content than either the control or the salt-evolved clones. Similarly, the thermally evolved clones stood out and slightly differed in metabolic profile by producing less lactate, acetate, and ethanol, and more pyruvate and formate than the unevolved ancestral control. The salt-evolved clone also produced less formate and ethanol, while producing more pyruvate than the control. The fatty acid composition also showed considerable variation between strains. Most notably, the thermally evolved clone BBi Heat 4.1, unlike both the other evolved clones and the control, had very little 16:0 fatty acid and was mostly unknown. BBi Heat 6.6 was quite similar to the control, while all the other evolved clones had less 18:1 and more unknown fatty acids.

[0090] Genetic changes All clones tested in the acceleration assay were submitted for whole genome sequencing. Because the clones had independent evolutionary histories, particular attention was paid to mutations that evolved in multiple strains, especially in cases where different independently evolved strains had acquired different mutations in the same gene.

[0091] Mutations in thermally evolved populations The thermally evolved clones with improved storage stability, Heat 4.1 and Heat 6.6, gained 12 and 13 mutations, respectively (see Examples). Seven of these mutations were present in the independently evolved strains, while two of the mutations were present in the same gene, but at different positions within the gene in the two evolved strains. We pay special attention to polyribonucleotide nucleotidyl transferase (EC 2.7.7.8), PNPase, and the response regulator consisting of a CheY-like receptor domain and a winged-helix DNA-binding domain, because we found mutations at different positions in both strains, suggesting convergent evolution.

[0092] Mutations in salt-evolved clones We sequenced two KCl-evolved and four NaCl-evolved clones. All mutations were synonymous SNPs, except for one nonsynonymous SNP in a putative glucose uptake permease and two mutations in an intergenic region. Three of those synonymous mutations were also present in the two thermally evolved clones (mutations in the arginine / ornithine antiporter ArcD, the pyridoxine biosynthesis glutamine aminotransferase glutaminase subunit, and the SSU ribosomal protein S5p (S2e)) but were not found when the ancestral strain was sequenced. The two KCl-evolved clones shared five additional mutations: SNPs in the intergenic regions of two hypothetical genes, a synonymous SNP in phosphoglucomutase, a nonsynonymous SNP for a stop codon in a hydrolase, and a nonsynonymous SNP in a hypothetical gene and in a response regulator consisting of a CheY-like receptor domain and a winged-helix DNA binding. The latter SNP was the same R175L mutation in both strains and was not found in the same gene in the thermally evolved clones. The NaCl evolved clones did not acquire any mutations in the gene. Among the four NaCl evolved clones, we observed 16 mutations present in all four clones. Five of them were synonymous SNPs and one mutation was in the intergenic region between the putative amidotransferase and the hypothetical gene. None of the salt evolved clones showed a consistent improvement in storage stability. KCl 1.2, NaCl 2.7, and NaCl 6.2 showed some improvement in storage stability. Four mutations were unique to NaCl 2.7 and NaCl 6.2. One of them was a synonymous change in a large transmembrane protein possibly involved in transport, and another mutation was in the intergenic region between the uridylyltransferase and a transcriptional regulator of the ArsR family. A deletion in the hypothetical gene probably caused a frameshift. The V58M mutation evolved in TrkA (a potassium uptake protein) in both independently improved the NaCl evolved clones.

[0093] An evolved strain of Lactobacillus paracasei (LCP37) Strain LCP37 evolved in the presence of several stressors including high temperature, oxgall, salts (KCl, NaCl), surfactants (SDS), and oxidants (peroxide). Two strains, the heat evolved clone and the SDS evolved clone, performed well after spot plating assays and were carried forward into the promotion assay.

[0094] Thermally evolved clones The results of the acceleration assay showed that the thermally evolved strains derived from the fermentation run at pH 5.2 demonstrated increased storage stability under dry conditions (50°C, no humidity), with the CFU and cell dry weight of the strains being comparable to the ancestral control.

[0095] Sequencing of the thermally evolved clones revealed 16 mutations, of which 9 were nonsynonymous, 4 were synonymous, and 3 were intergenic (Table A). Comparison of protein abundance of genes was performed in wild-type and thermally evolved clones. Of the 13 genes with mutations, proteomic data were generated for four genes: lagC_2, rpoB, htrA, and ICMKMEMM_01590 (aspartate aminotransferase). Only one of them, HtrA, was less abundant in evolved clones compared to wild-type controls at both pH=5.2 and pH=6.4.

[0096] [Table 2]

[0097] Although LPC37 was selected for increased resistance to heat for a fairly short time, it was possible to identify a single clone with almost 50% increased storage stability in dry conditions. The strain acquired only a few mutations, one of which was in tagatose-6-phosphatase (lacC_2). Tagatose was previously shown to improve cell survival during freezing. Therefore, mutations in the thermally evolved strain may increase the amount of tagatose in the cells, resulting in a more stable strain.

[0098] Evolution of Lactobacillus acidophilus NCFM (NCFM) Strains of NCFM were evolved against heat, NaCl, and KCl stressors. Preferred clones were selected based on the spot plating assay described above and then tested in the accelerated assay for improved stability. Thermally evolved clones were similar to the control for both CFU and cell dry weight. Storage stability of evolved clones was improved in both humid (30° C., 40% humidity) and dry (50° C., no humidity) conditions for both thermally evolved clones NCFM Heat 1.1 and NCFM Heat 6.3, and one KCl evolved clone, NCFM KCl 2.4. Two NaCl evolved clones, NCFM NaCl 1.2 and NCFM NaCl 3.2, were more storage stable in dry conditions.

[0099] The levels of intracellular organic acids at the end of fermentation were measured to provide an indication of the metabolic activity of the cells during fermentation. In general, differences were observed in the production of lactate, pyruvate, glycerol, formate, acetate, ethanol, and succinate between the unevolved and evolved strains in two replicate assays. Only acetate appeared to be slightly lower in the thermally evolved clones. This suggests that the metabolic activity of the evolved clones was comparable to the unevolved strains.

[0100] Bacterial fatty acid composition has been shown to be tightly linked to membrane fluidity and influences survival during freeze-drying (Louesdon et al. 2015). Overall, evolved cells are quite similar in fatty acid composition to the unevolved NCFM control.

[0101] To identify mutations that may be responsible for the observed improvement in storage stability, we sequenced each clone tested in the accelerated assay using whole genome sequencing and aligned the reads to the published reference sequence of L. acidophilus NCFM (Deatherage and Barrick 2014) using breseq. The two heat-evolved clones, NCFM Heat 1.1 and NCFM Heat 6.3, shared seven mutations, while two mutations were unique to NCFM Heat 6.3 (Tables 16 and 17, Example 31). Between the three KCl-evolved clones, we identified 18 different mutations (seven in NCFM KCl 1.6, 13 in NCFM KCl 2.4, and 10 in NCFM KCl 3.3), some of which were shared among the different strains (Table B). Similarly, we identified 16 unique mutations in the NaCl evolved clones (6 in NCFM NaCl 1.2, 10 in NCFM NaCl 3.2, and 5 in NCFM NaCl 6.4), and again, some of the mutations were shared between the different strains (Table C).

[0102] [Table 3]

[0103] [Table 4]

[0104] In summary, both thermally evolved clones carry an A232D mutation in clpC, which encodes the ATPase subunit of the ClpC-ClpP protease. This subunit directs proteins that are phosphorylated on arginine residues to ClpP for degradation and is involved in stress response. Proteomic analysis suggests that the mutation in the evolved strains appears to reduce the protein abundance of clpC, which may indicate a reduced stress response in the evolved strains. A stop codon (E14*) in gntR, which encodes a gntR family-like transcriptional regulator, has evolved in both strains, leading to a significant reduction in GntR. The gene gntR is part of the GntI operon. It represses the transcription of three other genes in the operon. In the presence of gluconate, GntR repression is reduced and three other genes involved in gluconate absorption are expressed. Also, in E. coli, the Entner-Doudoroff (ED) pathway is upregulated (Izu et al. 1997, Tsunedomi et al. 2003). However, L. acidophilus NCFM appears to lack two enzymes of the ED pathway. Thus, upregulation of the gnt operon due to lack of repression by GntR may be important for reducing oxidative stress or converting gluconate to ribose-5-phosphate. L. acidophilus NCFM has several genes annotated as gntR. EXAMPLES

[0105] The present invention is further defined in the following examples. It should be understood that these examples, while showing preferred embodiments of the present invention, are presented for illustrative purposes only. From the above discussion and these examples, a person skilled in the art can confirm the essential features of the present invention, and can make various changes and modifications of the present invention to suit various applications and conditions without departing from the spirit and scope of the present invention.

[0106] The abbreviations used are as follows: "min" means minute, "h" means hour, "μL" means microliter, "mL" means milliliter, "L" means liter, "nm" means "nanometer", "mm" means "millimeter", "cm" means "centimeter", "μm" means "micrometer", "mM" means "millimolar", "M" means "molar", "mmol" means millimole, "μmole" means micromole, "g" means gram, "mg" means milligram, "g" means gravitational constant, and "rpm" means revolutions per minute.

[0107] General method Materials and methods suitable for the maintenance and growth of microbial cultures are also well known in the art. Techniques suitable for use in the examples below can be found in Manual of Methods for General Bacteriology by Gerhardt et al. (Gerhardt et al., 1994) or in Biotechnology: A Textbook of Industrial Microbiology by Brock (Brock 1989). Yeast extract can be supplied by Angel (Yichang, China), Lesaffre (Marcq-en-Bareul, France), Ohly (Hamburg, Germany), DSM Food Specialties (Heerlen, Netherlands), Lallemand (Rexdale, Canada), Alltech Fermin (Nicholasville, KY), MC Food Specialties (Shinagawa-Ku, Japan), Yeastock (Tokyo, Japan), KOHJIN Life Sciences (Tokyo, Japan), Savoury Systems International (Branchburg, NJ), Sensient Technologies (Milwaukee, WI), or Kerry (Tralee, Ireland). All other reagents and materials used for the growth and maintenance of bacterial cells not specifically mentioned were obtained from Aldrich Chemicals (Milwaukee, WI), BD Diagnostic Systems (Sparks, MD), Life Technologies (Rockville, MD), or Sigma Chemical Company (St. Louis, MO) unless otherwise specified.

[0108] KK The probiotic strain Bifidobacterium animalis subspecies lactis Bi-07 (also called BBi) is a Gram-positive, rod-shaped, non-motile, anaerobic bacterium. The strain is available from ATCC as PTA SD5220. The strain has been sequenced and the sequence is available from NCBI under the accession number NC_017867 (Briczinski et al. 2009, Stahl and Barrangou 2012, Morovic et al. 2017). The sequence is characterized by a GC content of 60.5%.

[0109] Lactobacillus acidophilus NCFM is a Gram-positive, non-spore-forming bacterium isolated in the 1970s from a human source and subsequently characterized in the Food Microbiology Research Laboratory at North Caroline State University (Sanders and Klaenhammer 2001). The strain has been shown to have beneficial effects in humans when taken orally and has been sold commercially for over 25 years. The strain is available from ATCC as 700396. The genome has been fully sequenced and is available from NCBI under the accession number NC_006814.3 (Altermann et al. 2005). The GC content of the genome is 39.1%.

[0110] Experimental Evolution Strains evolved toward improved storage stability, essentially as described below. In general, repetitive populations were transferred serially into selective environments for about 100 generations, and single colonies were isolated and tested for improved storage stability. Strains with improved storage stability in spot plating assays were then tested in accelerated assays for fermentation performance, storage stability, metabolomics, fatty acids, and occasionally transcriptome changes, and proteomics profiles. Whole genome sequencing was performed to identify mutations. For example, single populations were grown and transferred into De Man, Rogosa, and Sharpe agar (MRS) broth, and then the populations were streaked on MRS agar and the plates were incubated at high temperature to select clones that could grow at high temperature. Alternatively, repetitive populations could be selected under a variety of conditions, such as:

[0111] Single colonies were isolated and stored to initiate replicate populations: frozen stocks were plated on MRS agar plates and grown under anaerobic conditions. Well-isolated colonies were picked and grown to high density in MRS broth, supplemented with glycerol, and frozen at -80°C. Single colony frozen stocks were subsequently used to initiate replicate populations in MRS broth supplemented with cysteine ​​HCl and stressors such as KCl, NaCl, SDS, or elevated temperature during incubation. All cultures were grown anaerobically in anaerobic boxes by covering the cultures with air-permeable film or by covering the cultures with aluminum foil. Agar plates were incubated in anaerobic boxes with the appropriate number of anaerobic GasPak sachets for the box size used.

[0112] Replicate selection experiments were performed as follows: typically, six or eight replicate populations were grown in 7 mL MRS broth supplemented with cysteine ​​(L-cysteine ​​HCl was added immediately before use at a final concentration of 0.5 g / L) at a starting OD of 0.2-0.3 and grown stationary or with slow (approximately 100 rpm) shaking for 24-48 hours before being transferred to fresh medium. Cultures were grown at 37°C in anaerobic conditions unless temperature was the stressor. The OD of several populations in the same selection regime was evaluated and populations were transferred at the same initial OD based on the average OD measured. When the populations reached an OD after 24 hours similar to the OD after 24 hours in mild conditions, the selection pressure was increased (i.e., the concentration of the stressor was increased or the temperature was increased). If the populations were unable to compensate for the dilution rate, the stressor was reduced. Samples of the population were frozen at regular intervals by supplementing the samples with glycerol and storing the samples at -80°C.

[0113] After approximately 100 generations, samples of the population were supplemented with glycerol and frozen at -80°C. The number of generations per transfer was calculated as follows:

number

[0114] Spot plating assay to identify storage-stable clones To assess the storage stability of evolved clones, single colonies from the evolved populations were isolated, grown in mild non-selective conditions, lyophilized, and evaluated against the ancestor for viability after up to 2 weeks in an environmental chamber. To isolate single colonies, evolved and ancestor populations were streaked or plated onto MRS agar plates and incubated with anaerobic GasPak sachets in an anaerobic box. The streaked populations were incubated for 2 days before isolating single colonies and restreaking onto fresh MRS agar plates, which were then incubated for another 2 days. To plate the populations onto MRS agar plates, cultures were serially diluted in 10-fold dilutions and plated onto MRS agar. After 2-3 days of incubation at 37°C in the anaerobic box, single, well-dispersed colonies were isolated and inoculated into fresh MRS broth in 48- or 96-well plates. Plates were sealed with clingy aluminum foil and incubated at 37°C, 100 rpm for 24 hours. The aluminum foil was carefully removed and the cells were resuspended by gently mixing the cultures with a multichannel pipettor or Viaflo96 (ViaFlo 96, Integra Biosciences AG, Zizers, Switzerland). The OD was measured and the cultures were diluted to OD 0.2 in 3.5 mL MRS broth in two new 48-well deep-well plates based on the average OD. The plates were sealed with adhesive aluminum foil and incubated overnight at 37°C, 100 rpm. At the same time, each clonal culture was supplemented with glycerol to bring the stocks to a final concentration of 20% glycerol and frozen at -80°C for further analysis. This process was performed on several single clones from replicate populations.

[0115] After incubation, cells were prepared for storage by flash freezing and lyophilization as follows: the aluminum seal was removed and cells were gently mixed into suspension in each well using a Viaflo96. The OD was measured and based on the average OD, cultures were added at OD4 in a volume equivalent to 1 mL to Cube2ubes (CUBE 2UBE® Tubes, Sigma BAF37851100) in a rack (CUBE 2UBE® 96-well rack with cover, Sigma BAF378560000) in a 96-well array. At least four Cube2ubes per culture were prepared to allow evaluation of the viability of clones at various time points. Cells were chilled on ice for 30 min and then centrifuged at 4° C., 3500 rpm for 10 min in a Multifuge X3R (ThermoFisher). All but 50 μL of supernatant was removed using a ViaFlo96, and 8 μL of potassium phosphate stabilizer (8 g monopotassium phosphate and 10.2 g dipotassium phosphate per 100 mL) was added to each Cube2ube and thoroughly mixed with the cells by shaking the cultures at 2000 rpm for 20 min in a Thermomixer C. After leaving the cells to rest for 10 min at room temperature and briefly mixing at 2000 rpm for 15 s, the Cube2ube were transferred in a box into liquid nitrogen for flash freezing and then placed in a freeze dryer for 24-72 h to freeze dry. The cubes were then placed in an environmental chamber and stored open (without cover) at 30°C and 40% humidity. The viability of the stored cells was assessed at regular intervals and compared to the viability of cells that were freeze dried but not stored as follows: one set of freeze dried cultures, the pre-storage set, was plated after freeze drying. All plating was performed by resuspending the cells in 210 μL MRS, mixing the cells at 2000 rpm for 20 seconds, allowing them to settle at room temperature for 30 minutes, mixing again at 2000 rpm for 15 seconds, and then diluting the culture in a 10-fold titration series. Add 5 μL of the particular dilution (e.g., 10 -6 , 10 -4 , 10 -2 , and 10 0) were spotted onto MRS agar plates in Fisher's OmniTrays (OmniTray Single Well w / Lid, Sterile PS 242811). After drying of the plated spots, the plates were placed in an anaerobic box with GasPak anaerobic sachets and incubated at 37°C for 2-3 days. After incubation, the highest dilution at which clones were able to grow was recorded and the plates were photographed. Similarly, another set was removed, resuspended, spotted and plated. The dilutions for plating were selected based on expected viability. Cultures were plated at different time intervals, typically after 0, 96, 168, 240 or 320 hours in the environmental chamber.

[0116] Clones with good survival rates in the spot plating assay were then tested in more detail in the accelerated assay and sequenced using whole genome sequencing to identify mutations that evolved during the selection experiment. To maintain evolutionary independence, improved clones from different populations were taken and tested in the accelerated assay. This approach allowed for the identification of potentially beneficial mutations using a comparative approach based on parallel evolution (Saxer et al. 2014). Storage stability in the accelerated assay was tested under dry conditions (50°C, no humidity), humid conditions (30°C, 40% humidity), and long-term storage (4 months) at 30°C and low humidity. Freezer vial lots were produced with every clone tested in the spot plating assay by growing the strain and freezing it after supplementation with 20% glycerol.

[0117] Bioreactor Culture The fermentation system was custom-built and consisted of twelve 350 mL jacketed CelStir spinner flasks (Wheaton Science Products, Millville, NJ), each equipped with an autoclavable glass probe (Cole-Parmer, Vernon Hills, IL) and an IKA ColorSquid magnetic stirrer (Cole-Parmer, Vernon Hills, IL). Each of the four vessels was temperature controlled by an Isotemp 4100 R20 Circulator (Thermo Fisher Scientific, Waltham, MA). A Eutech Alpha pH 200 controller (Thermo Fisher Scientific, Waltham, MA) was attached to a BioChem Valve micropump (Biochem Fluidics, Boonton, NJ) to control pH with a 20 μL dispense volume. pH and base addition were recorded in LabVIEW (National Instruments, Austin, TX) via a LabJack T7 Pro data acquisition unit (DAQ) (LabJack, Lakewood, Colorado). The fermentation settings are listed in Table 1. After the fermentation run was completed, the pH set point in the fermenter was changed to a neutral pH and the temperature set point to the "temperature after neutralization" value, and the culture was maintained at this condition for 1 hour.

[0118] [Table 5]

[0119] Culture medium The composition of the media used for shake flask experiments and for bioreactor cultivation of the strains is shown in Table 2. Water was taken from a public water utility (Experimental Station, Wilmington, DE). The media were adjusted to pH = 6.8 with 50% NH4OH solution for BBi and NCFM and 20% NaOH solution for LPC37, respectively, before autoclaving. The media were autoclaved in 800 mL aliquots contained in 1 L glass bottles (Corning Pyrex 1399-1000) at 121 °C for 20 min in an Amsco 3043 Jacketed Steam Sterilizer / Autoclave (Steris, Mentor, OH). For BBi, cysteine-HCl was added to the media after heat sterilization.

[0120] Seed culture The seed train of NCFM® included three steps: 1 mL of frozen stock culture was inoculated into a 15 mL Falcon™ tube filled with 14 mL of NCFM® medium (Table 2). The tube was capped and incubated in an Innova 4230 incubator (New Brunswick Scientific, Enfield, CT) at 38°C without agitation for 8 hours until an OD600 value of approximately 1.000 was reached. For the second stage, 100 mL of inoculum medium (Table 2) was inoculated in a 125 mL Fisherbrand flat-bottom closed-cap flask (Thermo Fisher Scientific, Waltham, MA), namely seed flask 2, and then incubated in an Innova 4230 incubator (New Brunswick Scientific, Enfield, CT) at 38°C without agitation for 16 hours. The target OD after inoculation was 0.078. At the end of the incubation time in seed flask 2, the pH was measured and only cultures with a pH of 4.0-4.4 were used for the next transfer. The inoculum volume for the third transfer was calculated for 100 mL medium (Table 2) in a 125 mL flat-bottom flask (seed flask 3). The target OD600 was 1.766. At the end of the incubation, if the pH was found to be above 4.4 or the OD was below 3, the culture was given additional time to grow to reach a pH of 4.0-4.4 and an OD of 3-4 before being transferred into the bioreactor. OD measurements were performed at 600 nm (OD600) with a Helios Alpha spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

[0121] The seed train of BBi included three steps. A 15 mL Falcon™ tube containing 14 mL of BBi medium (Table 2) was inoculated with 1 mL of frozen stock culture. The Falcon™ tube was capped and incubated for 8 hours at 38°C without agitation in an Innova 4230 incubator (New Brunswick Scientific, Enfield, CT). In the second stage, 100 mL medium (Table 2) was inoculated in a 125 mL Fisherbrand flat-bottom closed-cap flask (Thermo Fisher Scientific, Waltham, MA) to a target OD600 of 0.078. The flask was then incubated for 16 hours at 38°C without agitation in an Innova 4230 incubator (New Brunswick Scientific, Enfield, CT). At the end of the incubation, the pH was measured and only cultures with a pH of 4.0-4.4 were used for transfer into seed flask 3. The target OD was 1.766 in a 125 mL flat-bottom flask filled with 100 mL medium (Table 2). At the end of the incubation, if the pH was found to be above 4.4 or the OD was below 3, the culture was given additional time to grow to reach a pH of 4.0-4.4 and an OD of 3-4 before being transferred into the bioreactor. OD measurements were performed at 600 nm (OD600) with a Helios Alpha spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

[0122] [Table 6]

[0123] Analysis method OD600 was determined with a Helios Alpha spectrophotometer (P / N 9423 UVA 1202E, Thermo Fisher Scientific, Waltham, MA). sampleIf the value of OD exceeded 0.3, the culture was diluted with 0.9% NaCl using a dilution factor DF below 0.3. The optical density OD of the cell-free broth was measured after spinning 1 mL of culture at 16000 rpm for 2 min in an Eppendorf tabletop centrifuge (Eppendorf, Germany). broth Finally, the OD600 was calculated according to: O.D. 600 =DF×(A sample -A Nacl )-(OD broth -A Nacl )

[0124] To determine the cell dry weight concentration, a culture volume V of 2 or 3 mL (depending on the cell concentration (i.e., for OD600>10, 2 mL was chosen, and for OD600<10, 3 mL was chosen) was used. filtered was filtered through a Nylaflo filter (pore size 0.25 μm, diameter 47 mm, VWR Cat No 28140-040) placed on a 47 mm diameter fritted disk of a Synthware Filtration Apparatus (VWR cat# 60002-266, Kemtech America, Pleasant Prairie, WI) with a [ST]40 / 35 joint connection. The filter together with the filter cake was then washed with a 0.9% NaCl solution with a volume equal to the volume of the filtered culture, transferred to an aluminum pan, and placed in an oven to dry at 80° C. for 24 hours. The weight of the dried filter and filter cake in the aluminum pan, w dry The weight was calculated by subtracting the original weight of the filter and pan, w0, which was calculated before the experiment. In addition, the weight was calculated by subtracting the weight difference, w0, before and after drying of the reference filter that was simply washed with 0.9% NaCl solution. rf The concentration of cell dry weight cdw was determined according to:

number

[0125] Usually, cell dry weight concentrations were derived from the average of three independent measurements.

[0126] The concentration of colony forming units, CFU, in the cultures was determined usually at the end of the fermentation run (EOR) and / or after a "conditioning" process called "cold neutralization" (CN). 2 mL of EOR or CN cultures were transferred into 5 mL Eppendorf tubes and sonicated with a Q125 sonicator (Qsonica, Newtown, CT) with the Cl-18 probe centered in the tube, half-immersed in the culture medium, and operated at 40% amplitude for 15 seconds.

[0127] Sonicated culture samples were serially diluted in sterile 0.1% peptone water in 99 mL Flip-Top Dilution Bottles (3M, St. Paul, MN) and poured in triplicate with MRS agar for plating. MRS agar was usually prepared 1 week to 10 days before plating. Briefly, 70 g of Difco Lactobacilli MRS broth with agar (Becton, Dickinson and Company, Franklin Lakes, NJ) was added to 1 L of ultrapure water, mixed, and boiled for 1 min. 200 mL of MRS agar solution was aliquoted into 250 mL bottles and sterilized in a 20 min autoclave liquid cycle before being stored in a 48 C incubator until use. On the day of the experiment, the bottles were transferred to a water bath regulated at 46 °C. If the cells required cysteine ​​(e.g., BBi or NCFM), 2 mL of a 5% cysteine-HCl solution was added to the 200 mL MRS agar solution. The cysteine-HCl solution was prepared by dissolving cysteine-HCl powder in ultrapure water to 250 mL and then filter-sterilizing the solution with a 250 mL Nalgene aPES filter unit (55 mm diameter, 0.2 μm pore size) (Thermo Fisher Scientific, Waltham, MA). After allowing the plates to solidify for approximately 10 minutes at RT in an aerobic environment, the plates were inverted and stored in standard (up to 18 plates) or large (up to 33 plates) airtight containers (GasPak EZ Container System, Becton, Dickinson and Company, Franklin Lakes, NJ) with activated anaerobic pouches (GasPak EZ Container System Sachets, Becton, Dickinson and Company, Franklin Lakes, NJ) to create an oxygen-free atmosphere. The anaerobic containers were incubated for 44 hours in an environmental chamber set at 38°C.Plates of different dilutions were usually counted manually or occasionally based on processing of images generated by a Flash & Grow colony counter (IUL Instruments, Barcelona, ​​ES) with the image software Flash & Grow 1.3.

[0128] Downstream processing in 96-well "small-scale" plate assays Approximately 1 mL of the 200 mL culture broth taken directly from the bioreactor (which was then pH adjusted and stored in a 10° C. water bath for 1 h ("conditioning")) was transferred into tubes of a 96-plate (Nova Biostorage, Canonsburg, PA) by a Nimbus 96-head robot (Hamilton Robotics, Bonaduz, Switzerland) using a 1 mL Axygen tip (Thermo Fisher Scientific, Waltham, MA). A minimum of 3, usually 8 tubes per plate and condition were filled. The number of plates was chosen according to the stability testing program being performed, usually 5 plates. The filled plates were centrifuged at 4000 rpm at 10° C. for 10 min in an Eppendorf 5810 R centrifuge with an A-4-62 rotor. The supernatant was then removed from each tube by a LiHA (8-channel) Freedom EVO robot (Tecan, Maennedorf, Switzerland) using a liquid sensing / conductive 1000 μL tip (Thermo Fisher Scientific, Waltham, MA). Finally, stabilizer was added using a MCA 200 μL tip (Thermo Fisher Scientific, Waltham, MA). The stabilizer solution was prepared within 24 hours prior to the experiment. The typical amounts of supernatant removed and stabilizer added for strains BBi, BBl, LPC37, and NCFM® are shown in Tables 3 and 4, respectively.

[0129] [Table 7]

[0130] [Table 8]

[0131] The cell pellet and stabilizer solution were mixed in a V-shaped stirring element (V&P Scientific, San Diego, CA) and magnetic tumbler (V&P Scientific, San Diego, CA) at 200 rpm for approximately 2 minutes. The tubes of the plate were then transferred to a 96-well aluminum microplate frame (SP Scientific, Gardiner, NY) that had previously been stored on dry ice and then in a -80°C freezer. The aluminum frame with the tubes was lyophilized in a VirTis Advantage freeze dryer (SP Scientific, Gardiner, NY) according to the program shown in Table 5.

[0132] [Table 9]

[0133] After completion of the program, the tubes were transferred from the aluminum microplate frame into a plastic box. Plates for cell number determination without storage ("T0") were immediately transferred to a desiccator with a relative humidity of <1% and stored at room temperature for 1 hour before further processing.

[0134] Storage stability testing in a 96-well "mini-plate" assay at 30°C and 40% relative humidity After completion of the lyophilization program, the tubes were transferred from the aluminum microplate frame into a plastic box. For storage stability testing in the 96-well "mini-scale" plate assay at 30°C and 40% relative humidity, the plastic box with the open tubes was transferred into a model 7000-33-1 or 6040-1 Caron environmental chamber (Caron, Marietta, Ohio) set at 30°C and 40% relative humidity for storage until sampling.

[0135] Storage stability testing in a 96-well "mini-plate" assay at 50°C and low relative humidity After completion of the lyophilization program, the tubes were transferred from the aluminum microplate frame into a plastic box. For storage stability testing in the 96-well "mini-scale" plate assay at 50°C and low relative humidity, the plastic box with open tubes was transferred into a desiccator with a relative humidity of <1% and stored at 50°C until sampling.

[0136] Storage stability testing in a 96-well "mini-plate" assay at 30°C and low relative humidity After completion of the lyophilization program, the tubes were transferred from the aluminum microplate frame into a plastic box. For storage stability testing in the 96-well "mini-scale" plate assay at 30°C and low relative humidity, the plastic box with open tubes was transferred into a desiccator with low relative humidity <1% and stored at 50°C until sampling.

[0137] Cell revival and plating in 96-well "mini-plate" assays 1 mL MRS medium was added to each sample tube by a Nimbus 96-head robot (Hamilton Robotics, Bonaduz, Switzerland). The filled tubes (still containing the stir bar) were then agitated with a magnetic tumbler (V&P Scientific, San Diego, CA) at 300 rpm for 25 min, then at 600 rpm for another 5 min. Meanwhile, 900 μL peptone water was added into a 7×96-Corning well clear V-bottom 2 mL polypropylene deep-well plate #3960 (Corning Inc., Corning, NY) using a Nimbus 96-head robot (Hamilton Robotics, Bonaduz, Switzerland). After mixing, serial dilutions were generated by repeatedly transferring 100 μL sample volumes into 900 μL peptone water and then aspirating using a Nimbus 96-head robot. For each sample, the three most significant dilutions were usually selected for each dilution. Typically, three samples of variable volume, 50-200 uL each, were transferred onto a plate, MRS agar was poured on top, and the solution in the plate was thoroughly mixed. MRS agar was usually prepared 1 week-10 days before plating. Briefly, 70 g of Difco Lactobacilli MRS broth with agar (Becton, Dickinson and Company, Franklin Lakes, NJ) was added to 1 L of ultrapure water, mixed, and boiled for 1 min. 200 mL of MRS agar solution was aliquoted into 250 mL bottles, sterilized in a 20 min autoclave liquid cycle, and then stored in a 48 C incubator until use. On the day of the experiment, the bottles were transferred to a water bath regulated at 46 C. If the cells required cysteine ​​(e.g., BBi or NCFM), 2 mL of 5% cysteine-HCl solution was added to the 200 mL MRS agar solution.Cysteine-HCl solution was prepared by dissolving cysteine-HCl powder in ultrapure water to 250 mL and then filter-sterilizing the solution with a 250 mL Nalgene aPES filter unit (55 mm diameter, 0.2 μm pore size) (Thermo Fisher Scientific, Waltham, MA). After allowing the plates to cure for approximately 10 min at RT in an aerobic environment, the plates were inverted and stored in standard (up to 18 plates) or large (up to 33 plates) airtight containers (GasPak EZ Container System, Becton, Dickinson and Company, Franklin Lakes, NJ) with activated anaerobic pouches (GasPak EZ Container System Sachets, Becton, Dickinson and Company, Franklin Lakes, NJ) to create an oxygen-free atmosphere. The anaerobic containers were incubated for 44 h in an environmental chamber set at 38 °C. Plates of different dilutions were usually counted manually or occasionally based on processing of images generated by a Flash & Grow colony counter (IUL Instruments, Barcelona, ​​ES) with the image software Flash & Grow 1.3.

[0138] Statistical analysis of CFU counts Single Plate CFU count Since it is well established that high and low colony counts in the sham strain make the CFU results less accurate, a weighting function was introduced to estimate the CFU count >300 or CFU count gave results for samples with lower weights < 20: CFU count >300:w count =1 / [1+CFU count -300) / 50] 300 or more CFU count ≧25:w count =1 20>CFU count >0:w count =CFUcount / 20 CFU coun t = 0:w count = 0.02 Next, single outlier detection for all n CFU from the wells was performed for all dataset n > 3 based on the Dixon Q test. Depending on the number of suspected outliers, various ratios were used to identify potential outliers. Briefly, for x1 < x2 < … < xn-1 < xn and their corresponding ratios (r) - values for the lowest and highest values were determined as follows for n CFU count values x1~xn were ordered: plate

Number

[0139] Ratio determination was modified to ensure that the denominator xn - x1 is always at least 1% of x even for arrays showing minimal deviation in the measured values. If r(l) n or r(h) 10 exceeds the critical value r 10 (conf) 10 (conf) crit(number) is exceeded, the corresponding measured value was ignored in further analysis. The confidence interval was set to conf = 90%, and the respective critical value r(90%) crit(n) for the total number of measured values n per well / experiment was taken from Verma and Quiroz-Ruiz (Verma and Quiroz-Ruiz 2006). If both of the calculated values r(l) 10 and r(h) 10 exceed the critical value, only the measured value giving the larger r value was excluded. If eight or more observations were available per well, in addition to single outlier detection, the lowest and highest measured values were analyzed assuming both were outliers. Therefore, their r values were calculated as follows:

Number

[0140] ​The denominator xn-1-x1 or xn-x2 was again kept at a minimum of 1% of the next largest value xn-1. As in the procedure described above, r(l)11 or r(h)11 was determined with 90% confidence to be the critical value r11(conf) crit(number) (Verma and Quiroz-Ruiz 2006), the corresponding measurements were disregarded in the subsequent analysis. Overall, from n plates counted per well, the n' value passed the outlier test.

[0141] Finally, the weighted mean number of CFU per well, wCFUwell, and the associated weighted experimental standard deviation, wSDwell,exp, were calculated for each well according to:

number

number

[0142] Finally, the weights of the individual plate numbers were compiled into a quality score for the well by:

number

[0143] Results from all m wells representing the same condition were then pooled (at least 3, usually 8 wells per condition) and again subjected to the same Dixon-type outlier detection process as described above (yielding m' pass / fail values). The weighted mean CFU count per condition, wCFUcond, and associated weighted experimental standard deviation, wSDcond,exp, were calculated for each condition according to:

number

number

[0144] Error Propagation If the function was a nonlinear combination of a set of parameters, y=f(x1, x2, ..., xi), the function was linearized by approximating a first-order Taylor series expansion and error intervals calculated according to:

number

[0145] For example, the percent cell viability at a given time point T, SV(T), and its standard error of the mean, SEM(Sv(T)), were determined according to the following:

number

[0146] Whole genome sequencing All strains tested for storage stability in the accelerated assay were also analyzed at the genome level using whole genome sequencing. DNA was extracted using the MoBio PowerSoil kit and submitted for sequencing on a MiSeq. Paired-end reads were aligned to the reference sequence using the pipeline breseq (Deatherage and Barrick 2014). Both ancestral and evolved clones were sequenced. Only mutations present in the evolved clones were considered. Any mutations present in the ancestral strain were disregarded as sequencing errors in the reference sequence or differences between the ancestral and reference sequences.

[0147] Example 1 Adaptation to elevated heat results in increased storage tolerance for Bifidobacterium animalis subsp. lactis BBi Adaptation to heat results in a higher frequency of storage-stable clones than selection in medium alone. Recurrent populations of Bifidobacterium animalis subsp. lactis were evolved for approximately 100 generations in a variety of conditions, starting with a single clone, by daily transfer into fresh medium. One set of populations was evolved at a constant temperature of 37°C in De Man, Rogosa and Sharpe (MRS) broth supplemented with L-cysteine ​​HCl (Sigma C1276, CAS 52-89-1) at a final concentration of 0.5 g / L, while a second set was evolved in the same medium but at elevated temperature. Populations were transferred by dilution to a starting OD of 0.2-0.3. After approximately 100 generations of selection, samples of the evolved populations were frozen at -80°C for further analysis. Two randomly selected populations from each treatment were serially diluted, plated on MRS agar plates, and incubated at 37°C for 2 days under anaerobic conditions. The ancestral strain was also grown from the freezer, diluted, plated on MRS agar plates, and incubated with the evolved populations. After incubation, 16 randomly selected single clones were isolated from each evolved population, and two sets of 16 single clones were isolated from the ancestral population (Figure 1). These clones were then grown in MRS medium. After growth, the cells were spun down, the supernatant was removed, KP stabilizer was added, and the cell pellets were flash frozen and lyophilized before exposure to 50°C and 0% humidity for 22 days. For each clone, after lyophilization and storage at 50°C, the number of viable cells was assayed by eluting the pellet in MRS medium, plating serial dilutions on MRS agar plates, and counting the number of colony forming units per plate, as described in General Methods.

[0148] Overall, the frequency of strains with increased survival during storage was higher within the thermally evolved populations than among the ancestral clones or among the MRS-evolved populations (Fig. 2 ).

[0149] Example 2 Experimental evolution of Bifidobacterium animalis subsp. lactis BBi Six replicate populations were evolved for growth at increasingly higher temperatures starting from a single clone of the probiotic strain Bifidobacterium animalis subsp. lactis BBi (Bi-07 DuPont - hereafter referred to as BBi) as described in General Methods.

[0150] Populations were inoculated into 24-well blocks covered with sticky aluminum foil at an initial OD of 0.3 in 7 mL MRS broth supplemented with cysteine ​​and incubated at 42°C and 100 rpm. With a few exceptions where incubation lasted up to 70 hours, populations were transferred daily and inoculated at the same initial OD. The number of generations per day was calculated (Lenski et al. 1991):

number

[0151] After six transfers grown at 42° C., the incubation temperature was increased to 44° C. and maintained at that temperature for the remainder of the selection experiment. At least once per week, samples of each population were supplemented with glycerol (20% final concentration) and frozen at −80° C. After approximately 100 generations of selection, populations were supplemented with glycerol and frozen at −80° C. for further evaluation.

[0152] All six parallel populations evolved in the same way, and the stressor was increased in all populations simultaneously. None of the populations died out.

[0153] Example 3 A small-scale evaluation of the evolution of Bifidobacterium animalis subsp. lactis Bi-07 After the selection experiment, applicants isolated single clones from the evolved populations and tested their storage stability with a spot plating assay as described in the general methods. The spot plating assay sacrifices precision for high throughput: instead of evaluating CFU, applicants focused on the log10 change in viability of lyophilized samples relative to the ancestor after various storage intervals. Single clones were isolated by plating the evolved populations on MRS agar plates. Clones were grown to high density and flash frozen after adding KP stabilizer to the cell pellets. After lyophilization, the dried cell pellets were stored in an environmental chamber at 30° C. and 40% humidity, and cell viability was evaluated after various storage times and compared to that of the ancestral control strain. For each population, applicants evaluated up to seven single clones.

[0154] Two clones from two independent populations evolved for increased heat tolerance consistently showed higher storage stability than their ancestors after 120 and 264 hours. These clones, designated BBi Heat 4.1 and BBi Heat 6.6, were evaluated in accelerated assays to test their fermentation performance and subsequent storage stability. At the same time, their genomes were sequenced to identify mutations that had arisen during the progression of the selection experiment.

[0155] Example 4 Performance of BBi Heat 6.6 Seed cultures of BBi Wildtype C1 and BBi Heat 6.6 were generated following the three-step seed culture protocol described in General Methods. 7 mL of the second stage seed culture was inoculated into 343 mL of BBi medium (Table 2). One vessel each was inoculated with BBi Wildtype C1 and BBi Heat 6.6. BBi Wildtype C1 was harvested after 17.93 hours and BBi Heat 6.6 after 17.81 hours. The final cell dry weight concentration of BI Wildtype C1 was 3 cells g / l, while BBi heat 6.6 strain was 7 g / l, showing the improvement due to evolution.

[0156] Cultures of BBi Wildtype C1 and BBi Heat 6.6 were harvested into 500 mL shake flasks, capped, and stored in a 10° C. water bath for 1 hour. The cell concentration determined after storage was approximately 1.175×10 wildtype cell concentration. 10 CFU / mL, and the cell concentration of the heat 6.6 strain was approximately 1.35 × 10 10 CFU / mL.

[0157] Example 5 BBi Heat 6.6 Heat Performance in Downstream Processing The sample material generated in Example 3 was processed according to the protocol under "Downstream Processing in 96-well "Small-Scale" Plate Assay" as described in General Methods. Along the processing steps, cell numbers were determined after the conditioning step as "CN" and a second time after downstream processing as "T0" as described in the General Methods section. Viability (downstream processing) was then calculated according to

number

[0158] The analysis showed that under the assay conditions specified, strain BBi Heat 6.6 demonstrated approximately 75% viability, whereas the BBi Wildtype C1 control only demonstrated approximately 50% viability.

[0159] Example 6 Storage stability of BBi Heat 6.6 Heat Sample material produced in Example 5 was stored at 30° C., 40% relative humidity according to the protocol described under "Storage stability testing by 96-well "mini-scale" plate assay at 30° C., 40% relative humidity" in General Methods. The material was then tested for CFU content after storage at 30° C., 40% relative humidity for 14 d (CFU(storage, 30C, wet, 14 d)) or 28 d (CFU(storage, 30C, wet, 28 d)) using the methods described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 30C, wet, X) at each storage time X was calculated according to the following:

number

[0160] Strain BBi Heat 6.6 was found to have significantly improved viability, i.e., significantly improved storage stability, when stored at 30°C and 40% relative humidity for 14 or 28 d compared to BBi Wildtype C1 (BBi Wildtype C1 showed approximately 7.5% viability at 14 and 287 days, while strain BBi Heat 6.6 demonstrated approximately 24% viability at 14 days and 20% viability at 28 days).

[0161] Sample material produced in Example 5 was stored at 50° C. at low relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 50° C. at low relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 50° C. at low relative humidity for 7 d (CFU(storage, 50C, dry, 7 d)) or 14 d (CFU(storage, 50C, dry, 14 d)) using the methods described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 50C, dry, X) at each storage time X was calculated according to the following:

number

[0162] Strain BBi Heat 6.6 was found to have significantly improved viability, i.e., significantly improved storage stability, when stored at 50°C and low relative humidity for 7 or 14 d compared to BBi Wildtype C1 (BBi Wildtype C1 showed 10% viability at 7 days and 5% at 14 days under the specified conditions, while strain BBi Heat 6.6 demonstrated 30% viability at 7 days and approximately 18% viability at 14 days under the specified conditions).

[0163] Example 7 Effective production capacity of BBi Heat 6.6 Heat under different storage conditions Information was obtained from culturing the strains as described in Example 4, processing the strains as described in Example 5, and analyzing the strains as described in the Examples to derive the effective productivity of the strains according to the following:

number

[0164] In this example, for ΔCFU, we took the surviving "equivalent" cell concentration for the strain in the last stage of fermentation. In this example, the cells provided in the inoculum were considered insignificant, and consequently, the "equivalent" cell concentration for the strain was not corrected. fermentation For the sake of convenience, only the fermentation time for the strains in the last stage vessel was considered. After downstream processing and storage of the cultures for 14 d or 28 d at 30° C. and 40% relative humidity, strain BBi Heat 6.6 was found to exhibit a significantly higher effective productivity than BBi Wildtype C1 ( FIG. 3 ).

[0165] Effective productivity determinations were performed. Strains were cultivated as described in Example 4, processed as described in Example 5, and stored at 50° C. and low relative humidity for 7 d or 14 d as described in Example 6. Strain BBi Heat 6.6 was found to exhibit significantly higher effective productivity than BBi Wildtype C1 ( FIG. 4 ).

[0166] Example 8 Performance of strains BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 Seed cultures of BBi Wildtype C1, BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were generated according to the three-step seed culture protocol described in the "General Methods" section. 7 mL of the second stage seed culture was inoculated into 343 mL of BBi medium (Table 2). One vessel each was inoculated with BBi Wildtype C1, BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2. BBi Wildtype C1 was harvested after 17.93 hours, BBi KCl 1.2 after 15.65 hours, BBi KCl 5.5 after 15.58 hours, BBi NaCl 2.7 after 15.55 hours, BBi NaCl 3.3 after 15.61 hours, BBi NaCl 5.1 after 15.59 hours, and BBi NaCl 6.2 after 15.59 hours. Final cell dry weight concentrations at harvest are shown in Figure 5.

[0167] Cultures of BBi Wildtype C1, and BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were harvested in 500 mL shake flasks, capped, and stored in a water bath at 10 C for 1 hour. The measured cell concentrations CFU / mL correlated strongly with the cell dry wt. data, as shown in FIG. 5.

[0168] Example 9 Performance of strains BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 in downstream processing The sample material generated in Example 8 was processed according to the protocol under "Downstream Processing in 96-well "Small-Scale" Plate Assay" described in the General Methods section. Along the processing steps, cell numbers were determined after the conditioning step as "CN" and a second time after downstream processing as "T0" as described in the General Methods section. Viability (downstream processing) was then calculated according to

number

[0169] As shown in FIG. 6, strains BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 showed higher stability in downstream processing than BBi Wildtype C1.

[0170] Example 10 Storage stability of strains BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 Sample material produced in Example 9 was stored at 30° C., 40% relative humidity according to the protocol described under "Storage stability testing by 96-well "mini-scale" plate assay at 30° C., 40% relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 30° C., 40% relative humidity for 14 d (CFU(storage, 30C, wet, 14 d)) or 28 d (CFU(storage, 30C, wet, 28 d)) using the methods described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 30C, wet, X) at each storage time X was calculated according to the following:

number

[0171] Strains BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, and BBi NaCl 6.2 were found to have significantly improved viability, i.e., storage stability, when stored for 14 or 28 d at 30° C. and 40% relative humidity, compared to BBi Wildtype C1. Strains BBi NaCl 3.3 and BBi NaCl 5.1 showed improved viability after 14 d of storage, but not after 28 d of storage (FIG. 7).

[0172] Sample material produced in Example 9 was stored at 50° C. at low relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 50° C. at low relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 50° C. at low relative humidity for 7 d (CFU(storage, 50C, dry, 7 d)) or 14 d (CFU(storage, 50C, dry, 14 d)) using the methods described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 50C, dry, X) at each storage time X was calculated according to the following:

number

[0173] Strains BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were found to have improved viability, i.e., improved storage stability, when stored for 7 or 14 d at 50° C. and low relative humidity, compared to BBi Wildtype C1. Strain BBi KCl 5.5 showed improved storage stability after 7 d of storage, but no longer at 14 d (data not shown).

[0174] Example 11 Effective productivity of BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 for various storage conditions Information was obtained from culturing the strain as described in Example 8, processing the strain as described in Example 9, and analyzing the strain as described in Example 10 to derive the effective productivity of the strain according to the following:

number

[0175] In this example, for ΔCFU, we took the surviving "equivalent" cell concentration for the strain in the last stage of fermentation. The cells provided in the inoculum in this example were considered insignificant, and consequently the "equivalent" cell concentration for the strain was not corrected. fermentation For the sake of convenience, only the fermentation time for the strains in the last stage vessel was considered. After downstream processing and storage of the cultures for 14 d or 28 d at 30° C. and 40% relative humidity, strains BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were found to show higher effective productivity than BBi Wildtype C1 ( FIG. 8 ).

[0176] Effective productivity determinations were performed. Strains were cultivated as described in Example 8, processed as described in Example 9, and stored at 50° C. and low relative humidity for 7 d or 14 d as described in Example 10. Strains BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were found to exhibit higher effective productivity than BBi Wildtype C1 (data not shown).

[0177] Example 12 Performance of BBi Heat 4.1 and BBi Heat 6.6 Heat Seed cultures of BBi Wildtype C1, BBi Heat 4.1, and BBi Heat 6.6 were generated following the three-step seed culture protocol described in the General Methods section. 7 mL of the second stage seed culture was inoculated into 343 mL of BBi medium (Table 2). One vessel each was inoculated with BBi Wildtype C1, BBi Heat 4.1, and BBi Heat 6.6. The cumulative base addition (Table 1) was recorded. The time course of cumulative base addition for the three fermentation runs is shown in Figure 9.

[0178] BBi Wildtype C1 was harvested after 18.25 hours, BBi Heat 4.1 after 18.95 hours, and BBi Heat 6.6 after 19.10 hours. Final cell dry weight concentrations at harvest demonstrated a reduction in concentration for both thermally evolved strains (cell concentrations were approximately 4.8 g / l and 5 g / l for strains BBi Heat 4.1 and BBi Heat 6.6, respectively, while the concentration for BBi Wildtype C1 was approximately 5.7 g / l). The change in harvest time may explain this negative result.

[0179] Cultures of BBi Wildtype C1, BBi Heat 4.1, and BBi Heat 6.6 were harvested into 500 mL shake flasks, capped, and stored in a 10 C water bath for 1 hour. Cell concentrations determined after storage demonstrated a similar decrease for the thermally evolved strains. Concentrations were approximately 8 x 10 for BBi Heat 4.1 and BBi Heat 6.6, respectively. 10 CFU / mL and 1×10 10 CFU / mL, whereas BBi Wildtype C1 was approximately 1.55 × 10 10 CFU / ml was demonstrated.

[0180] Example 13 Performance of BBi Heat 4.1 and BBi Heat 6.6 Heat in Downstream Processing The sample material generated in Example 12 was processed according to the protocol under "Downstream Processing in 96-well "Small-Scale" Plate Assay" described in the General Methods section. Along the processing steps, cell numbers were determined after the conditioning step as "CN" and a second time after downstream processing as "T0" as described in the General Methods section. Viability (downstream processing) was then calculated according to

number

[0181] Strains BBi Heat 4.1 and BBi Heat 6.6 showed greater stability in downstream processing than BBi Wildtype C1 (BBi Heat 4.1 and BBi Heat 6.6 showed approximately 82% and 75% viability, respectively, while BBi Wildtype C1 demonstrated approximately 68% viability).

[0182] Example 14 Storage stability of BBi Heat 4.1 and BBi Heat 6.6 Heat Sample material produced in Example 13 was stored at 30° C., 40% relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 30° C., 40% relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 30° C., 40% relative humidity ("wet") for 14 d (CFU (30C, Wet, 14d)) using the method described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 30C, wet, 14d) was calculated according to the following:

number

[0183] Strains BBi Heat 4.1 and BBi Heat 6.6 were found to have significantly improved viability, i.e., significantly improved storage stability, when stored at 30° C. and 40% relative humidity for 14 d compared to BBi Wildtype C1 (BBi Heat 4.1 and BBi Heat 6.6 had approximately 32% and 47% viability, respectively, while BBi Wildtype C1 demonstrated only about 10% viability).

[0184] Sample material produced in Example 13 was stored at 30° C. at low relative humidity according to the protocol described under "Storage stability testing by 96-well "mini-scale" plate assay at 30° C. at low relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 30° C. at low relative humidity for 137 d (CFU (30C, dry, 137 d)) using the method described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 30C, dry, 137 d) was calculated according to the following:

number

[0185] Strains BBi Heat 4.1 and BBi Heat 6.6 were found to have significantly improved viability, i.e., significantly improved storage stability, when stored at 30° C. and low relative humidity for 137 d compared to BBi Wildtype C1 (data correlated well with data for the 30° C., humid, 14 d assay described above).

[0186] Sample material produced in Example 13 was stored at 50° C. at low relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 50° C. at low relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 50° C. at low relative humidity for 14 d (CFU (50C, dry, 14 d)) using the method described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 50C, dry, 14 d) was calculated according to the following:

number

[0187] Strain BBi Heat 4.1 was found to have significantly improved viability, i.e., significantly improved storage stability, when stored for 14 d at 50° C. and low relative humidity, compared to BBi Wildtype C1. However, strain BBi Heat 6.6 demonstrated no improvement over BBi Wildtype C1, showing an approximately 50% reduction in viability.

[0188] Example 15 Effective production capacity of BBi Heat 4.1 and BBi Heat 6.6 Heat under various storage conditions Information was obtained from culturing the strain as described in Example 12, processing the strain as described in Example 13, and analyzing the strain as described in Example 14 to derive the effective productivity of the strain according to the following:

number

[0189] In this example, for ΔCFU, we took the surviving "equivalent" cell concentration for the strain in the last stage of fermentation. The cells provided in the inoculum in this example were considered insignificant, and consequently the "equivalent" cell concentration for the strain was not corrected. fermentationOnly the fermentation time for the strains in the last stage vessel was considered. After downstream processing and storage of the cultures for 14 d at 30° C. and 40% relative humidity, strains BBi Heat 4.1 and BBi Heat 6.6 were found to exhibit significantly higher effective productivity than BBi Wildtype C1 ( FIG. 10 ).

[0190] Effective productivity determinations were performed. Strains were grown as described in Example 12, processed as described in Example 13, and stored for 137 d at 30° C. and low relative humidity as described in Example 14. Strains BBi Heat 4.1 and BBi Heat 6.6 were found to exhibit significantly higher effective productivity than BBi Wildtype C1.

[0191] Effective productivity determinations were performed. Strains were cultivated as described in the Examples, processed as described in Example 13, and stored for 14 d at 50° C. and low relative humidity as described in Example 14. Strain BBi Heat 4.1 was found to exhibit significantly higher effective productivity than BBi Wildtype C1 ( FIG. 11 ).

[0192] Example 16 Performance of strains BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 Seed cultures of BBi Wildtype C1, BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were generated following the three-step seed culture protocol described in the General Methods section. 7 mL of the second stage seed culture was inoculated into 343 mL of BBi medium (Table 2). One vessel each was inoculated with BBi Wildtype C1, BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2. The cumulative base addition (Table 1) was recorded. The time course of cumulative base addition for the three fermentation runs is shown in FIG. 12.

[0193] BBi Wildtype C1 was harvested after 18.25 hours, BBi KCl 1.2 after 15.45 hours, BBi NaCl 2.7 after 15.45 hours, BBi NaCl 3.3 after 18.25 hours, BBi NaCl 5.1 after 15.35 hours, and BBi NaCl 6.2 after 15.35 hours. Final cell dry weight concentrations at harvest are shown in Figure 13.

[0194] Cultures of BBi Wildtype C1, BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were harvested into 500 mL shake flasks, capped, and stored in a water bath at 10 C for 1 hour. The cell concentrations determined after storage are shown in FIG.

[0195] Example 17 Performance of BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 in downstream processing The sample material generated in Example 16 was processed according to the protocol under "Downstream Processing in 96-well "Small-Scale" Plate Assay" described in the General Methods section. Along the processing steps, cell numbers were determined after the conditioning step as "CN" and a second time after downstream processing as "T0" as described in the General Methods section. Viability (downstream processing) was then calculated according to

number

[0196] As shown in FIG. 15, strains BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 5.1, and BBi NaCl 6.2 exhibited greater stability in downstream processing than BBi Wildtype C1.

[0197] Example 18 Storage stability of BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 Sample material from Example 17 was stored at 30° C., 40% relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 30° C., 40% relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 30° C., 40% relative humidity ("wet") for 14 d (CFU (30C, Wet, 14d)) using the method described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 30C, wet, 14d) was calculated according to

number

[0198] Strains BBi Heat 4.1 and BBi Heat 6.6 were found to have significantly improved viability, i.e., significantly improved storage stability, when stored at 30° C. and 40% relative humidity for 14 d compared to BBi Wildtype C1 (FIG. 16).

[0199] Sample material produced in Example 17 was stored at 30° C. at low relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 30° C. at low relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 30° C. at low relative humidity for 137 d (CFU (30C, dry, 137 d)) using the method described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 30C, dry, 137 d) was calculated according to the following:

number

[0200] Strains BBi KCl 1.2, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were found to have improved viability, i.e., improved storage stability, when stored at 30° C. and low relative humidity for 137 d compared to BBi Wildtype C1 (FIG. 17).

[0201] Sample material produced in Example 17 was stored at 50° C. at low relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 50° C. at low relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 50° C. at low relative humidity for 14 d (CFU (50° C., dry, 14 d)) using the method described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 50° C., dry, 147 d) was calculated according to the following:

number

[0202] It was found that none of the strains showed significantly improved viability, i.e., storage stability, when stored at 50° C. and low relative humidity for 14 d compared to BBi Wildtype C1 (FIG. 18).

[0203] Example 19 Effective productivity of BBi KCl 1.2, BBi NaCl 2.7, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 Information was obtained from culturing the strain as described in Example 16, processing the strain as described in Example 17, and analyzing the strain after storage as described in Example 18 to derive the effective productivity of the strain according to the following:

number

[0204] In this example, for ΔCFU, we took the surviving "equivalent" cell concentration for the strain in the last stage of fermentation. The cells provided in the inoculum in this example were considered insignificant, and consequently the "equivalent" cell concentration for the strain was not corrected. fermentation For the sake of convenience, only the fermentation time for the strains in the last stage vessel was considered. After downstream processing and storage of the cultures for 14 d at 30° C. and 40% relative humidity, strains BBi NaCl 2.7, BBi NaCl 3.3, and BBi NaCl 6.2 were found to exhibit significantly higher effective productivity than BBi Wildtype C1 ( FIG. 19 ).

[0205] Effective productivity determinations were performed. Strains were grown as described in Example 16, processed as described in Example 17, and stored for 137 d at 30° C. and low relative humidity as described in Example 18. Strains BBi KCl 1.2, BBi NaCl 3.3, BBi NaCl 5.1, and BBi NaCl 6.2 were found to exhibit significantly higher effective productivity than BBi Wildtype C1 ( FIG. 20 ).

[0206] Effective productivity determinations were performed. Strains were grown as described in Example 16, processed as described in Example 17, and stored for 14 d at 50° C. and low relative humidity as described in Example 18. None of the strains were found to show significantly higher effective productivity than BBi Wildtype C1 ( FIG. 21 ).

[0207] Example 20 Genetic changes in thermally and salt-evolved BBi clones Applicants sequenced the two heat-evolved and six salt-evolved clones (BBi Heat 4.1, BBi Heat 6.6, BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3., BBi NaCl 5.1, and BBi NaCl 6.2) tested in the accelerated assay using whole genome sequencing and aligned reads to the reference sequence of the ancestral BBi clone (NC_017867) using breseq (see General Methods for details). To determine whether mutations evolved during the progression of the selection experiment, Applicants also sequenced the ancestral strain to identify differences between the reference sequence and the ancestral strain. For further analysis, only mutations that were not present in the ancestor but present in the evolved clones were considered. Mutations present in the ancestral strains were considered most likely to be sequencing errors in the reference sequence or differences between the ancestral strain and the reference strain that was originally sequenced. Because the clones have independent evolutionary histories due to their isolation from different populations, we were particularly interested in mutations that evolved in multiple strains, especially when genes acquired different mutations in different strains. Such parallel evolution often represents beneficial or selective mutations.

[0208] The heat-evolved clones with improved storage stability, BBi Heat 4.1 and BBi Heat 6.6, acquired 12 and 13 mutations, respectively (Table 6, Table 7). Seven of these mutations were present in both independently evolved strains. Two genes, polyribonucleotide nucleotidyl transferase (EC 2.7.7.8) and a response regulator consisting of a CheY-like receptor domain and a winged-helix DNA-binding domain, are of particular interest because they acquired mutations at different sites within the genes, suggesting that the mutations may be due to convergent evolution and that these genes may be targets of selection. Some of the mutations in these heat-evolved strains were additionally found in the salt-evolved BBi strain, indicating greater significance.

[0209] [Table 10]

[0210] [Table 11]

[0211] Also, although not all clones from a population had the same mutations, mutations in the PNPase or response regulator consisting of a CheY-like receptor domain and a winged-helix DNA-binding domain are present in other clones from the same population, and it is reasonable to conclude that mutations in these genes are beneficial for adaptation to the selection conditions.

[0212] Two KCl evolved clones and four NaCl evolved clones were sequenced as described in General Methods. Eight mutations were present in all six clones. The two KCl evolved clones shared five mutations (one in the intergenic region between the two hypothetical genes, one SNP for the stop codon in the hydrolase, and two nonsynonymous SNPs in the hypothetical gene, as well as a substitution in the response regulator consisting of the receptor domain and winged helix DNA binding for CheY). Both KCl evolved strains shared the same R175L mutation in the response regulator consisting of the receptor domain and winged helix DNA binding for CheY, while differing from the two mutations in this gene observed in the thermally evolved clone. The NaCl evolved clone did not acquire any mutations in this gene. Of the four NaCl evolved clones, 16 mutations were observed to be present in all four clones, five of which were synonymous SNPs, and one mutation was in the intergenic region between the putative amidotransferase and the hypothetical gene. Four mutations were unique to NaCl 2.7 and NaCl 6.2. One of them was a synonymous change in a large transmembrane protein likely involved in transport. Another mutation was in the intergenic region between a uridylyltransferase and a transcriptional regulator of the ArsR family. A deletion in a hypothetical gene likely caused a frameshift. In both cases, the evolved V58M mutation in TrkA (a potassium uptake protein) independently improved the NaCl evolved clone.

[0213] Mutations found in BBi KCl 1.2, BBi KCl 5.5, BBi NaCl 2.7, BBi NaCl 3.3., BBi NaCl 5.1, and BBi NaCl 6.2 are found in Tables 8-13, respectively. Mutations common to all KCl evolved BBi strains are found in Table 14. Mutations common to all NaCl evolved strains are found in Table 15. Mutations common to all salt evolved BBi strains are found in Table 16.

[0214] [Table 12]

[0215]

Table 13

[0216]

Table 14

[0217]

Table 15

[0218]

Table 16

[0219]

Table 17

[0220]

Table 18

[0221]

Table 19

[0222]

Table 20

[0223]

Table 21

[0224]

Table 22

[0225] [Table 23]

[0226] [Table 24]

[0227] Example 21 Testing the stability of evolutionary mutations in BBi Heat 4.1 and BBi Heat 6.6 during fermentation To test whether the new mutation was stably maintained in the clonal population during fermentation and production, applicants sequenced the end of the run population from small-scale fermentation AA029 to check for polymorphism of the new mutation in the population. Such polymorphism would indicate new mutations or reversions occurring during the fermentation process. Since the process started with a clonal population, it was expected that all evolutionary mutations would occur at a frequency of 1 (100%) in the population. If the mutation was not stable, the frequency would be expected to be less than 1.

[0228] Six populations from the EOR fermentation were sequenced and the populations were analyzed as polymorphic populations with breseq (Deatherage and Barrick 2014) using the polymorphism option (-p). This analysis confirmed that the mutations found in the EOR samples from the fermentation runs with this clone, and all mutations, were present at 100% frequency in the populations, suggesting that revertants had not evolved at measurable frequencies.

[0229] Example 22 Evolution and selection of Lactobacillus acidophilus NCFM Experimental evolution and identification of improved clones were performed as described in General Methods. Briefly, eight single colonies were isolated by streaking frozen L. acidophilus NCFM stock MRS agar. These colonies were grown in MRS agar and then used to initiate eight replicate populations that were selected in different selective environments. Samples of these cultures were supplemented with 20% glycerol and frozen at -80°C.

[0230] Eight parallel populations were initiated by inoculating 7 mL MRS supplemented with cysteine ​​in 24-well plates at a starting OD of 0.2, covered with clingy aluminum foil and incubated for 24 h at 42°C, 100 rpm. Populations were transferred to fresh medium daily, with a few exceptions that were transferred after a maximum of 48 h of incubation. At the time of transfer, 7 mL MRS with cysteine ​​was inoculated with cultures at an OD of 0.2. The number of generations per transfer was calculated as follows (Lenski et al. 1991):

number

[0231] Populations were transferred daily as described above. After 6 days, the temperature was increased to 44° C. for the remainder of the selection experiment. At least once per week, samples of each population were supplemented with glycerol (20% final concentration) and frozen at −80° C. All selections were run for approximately 100 generations. After the experiment was completed, samples of each population were supplemented with 20% glycerol and frozen at −80° C. for further analysis.

[0232] To evaluate the storage stability of the thermally evolved clones, five single colonies were isolated as described in General Methods. These single clones were then grown under mild conditions at 37°C, 100 rpm for 24 hours under anaerobic conditions. The cultures were transferred at an OD of 0.2 to 3.5 mL MRS supplemented with cysteine ​​in two new 48-deep-well plates and incubated overnight at 37°C, 100 rpm. At the same time, each clone culture was supplemented with glycerol to make the stocks a final concentration of 20% glycerol and frozen at -80°C. After incubation, 1 mL cultures at OD=4 were spun down, the supernatant was removed, stabilizer was added, and the pellets were flash frozen and lyophilized in a freeze dryer for 24 hours, as described in the General Methods section. After lyophilization for 24 hours, the pellets were stored at 30°C and 40% humidity and the viability of the clones was evaluated at regular intervals: immediately after lyophilization, 96, 196, and 290 hours after storage. Cultures were resuspended in MRS broth supplemented with cysteine, serially diluted 10-fold, and 5 μl of the dilutions were spotted on MRS agar plates. Most populations had clones with improved storage stability when compared to the ancestor. Two clones from independently evolved populations, NCFM Heat 1.1 and NCFM Heat 6.3, were selected for further investigation.

[0233] Example 23 Performance of strains NCFM Heat 1.1 and NCFM Heat 6.3 Seed cultures of NCFM Wildtype C1, NCFM Wildtype C2, NCFM Heat 1.1, and NCFM Heat 6.3 were generated following the three-step seed culture protocol described in the General Methods section. 7 mL of second stage seed culture was inoculated into 343 mL of NCFM medium (Table 2). One vessel each was inoculated with NCFM Wildtype C1, NCFM Wildtype C2, NCFM Heat 1.1, and NCFM Heat 6.3. Cumulative base addition (Table 1) was recorded. The time course of cumulative base addition for the four fermentation runs is shown (Figure 22).

[0234] NCFM Wildtype C1 was harvested after 13.63 hours, NCFM Wildtype C2 after 14.73 hours, NCFM Heat 1.1 after 14.73 hours, and NCFM Heat 6.3 after 14.73 hours. Final cell dry weight concentrations at harvest are shown in Figure 23. Cultures of NCFM Wildtype C1, NCFM Wildtype C2, NCFM Heat 1.1, and NCFM Heat 6.3 were harvested in 500 mL shake flasks, capped, and stored in a 10 C water bath for 1 hour. Cell concentrations determined after storage are shown in Figure 24.

[0235] Example 24 Performance of strains NCFM Heat 1.1 and NCFM Heat 6.3 in downstream processing The sample material generated in Example 23 was processed according to the protocol under "Downstream Processing in 96-well "Small-Scale" Plate Assay" described in the General Methods section. Along the processing steps, cell numbers were determined after the conditioning step as "CN" and a second time after downstream processing as "T0" as described in the General Methods section. Viability (downstream processing) was then calculated according to

number

[0236] Example 25 Storage stability of strains NCFM Heat 1.1 and NCFM Heat 6.3 Sample material produced in Example 24 was stored at 30° C., 40% relative humidity according to the protocol described under "Storage stability testing by 96-well "mini-scale" plate assay at 30° C., 40% relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 30° C., 40% relative humidity for X=14 d (CFU(storage, 30C, wet, 14 d)) or X=28 d (CFU(storage, 30C, wet, 28 d)) using the methods described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 30C, wet, X) at each storage time X was calculated according to the following:

number

[0237] Both strains, NCFM Heat 1.1 and NCFM Heat 6.3, were found to have significantly improved viability, i.e., significantly improved storage stability, when stored at 30° C. and 40% relative humidity for 14 or 28 d compared to the two control cultures, NCFM Wildtype C1 and NCFM Wildtype C1 (FIG. 26).

[0238] Sample material produced in Example 24 was stored at 50° C. at low relative humidity according to the protocol described under “Storage stability testing by 96-well “small-scale” plate assay at 50° C. at low relative humidity” in the General Methods section. The material was then tested for CFU content after storage at 50° C. at low relative humidity for X=7 d (CFU(storage, 50C, dry, 7 d)) or X=14 d (CFU(storage, 50C, dry, 14 d)) using the methods described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 50C, dry, X) at each storage time X was calculated according to the following:

number

[0239] Strain NCFM Heat 1.1 was found to have significantly improved viability, i.e., significantly improved storage stability, when stored at 50° C. and low % relative humidity for 7 or 14 d compared to both control strains, NCFM Wildtype C1 and NCFM Wildtype C2. However, only strain NCFM Heat 6.3 showed improved storage stability compared to the control culture NCFM Wildtype C2, but less storage stability than the NCFM Wildtype C1 culture, when stored at 50° C. and low % relative humidity for 7 or 14 d (FIG. 27).

[0240] Example 26 Effective production capacity of NCFM Heat 1.1 Heat and NCFM Heat 6.3 under different storage conditions Information was obtained from culturing the strain as described in Example 23, processing the strain as described in Example 24, and analyzing the strain as described in Example 25 to derive the effective productivity of the strain according to the following:

number

[0241] In this example, for ΔCFU, we took the surviving "equivalent" cell concentration for the strain in the last stage of fermentation. The cells provided in the inoculum in this example were considered insignificant, and consequently the "equivalent" cell concentration for the strain was not corrected. fermentation For the sake of convenience, only the fermentation time for the strains in the last stage vessel was considered. After downstream processing and storage of the cultures for 14 d or 28 d at 30° C. and 40% relative humidity, both evolved strains NCFM Heat 1.1 and NCFM Heat 6.3 were found to exhibit significantly higher effective productivity than both control strains NCFM Wildtype C1 and NCFM Wildtype C2 ( FIG. 28 ).

[0242] Effective productivity determinations were performed. Strains were cultured as described in Example 23, processed as described in Example 24, and stored for 7 d or 14 d at 50° C. and low relative humidity as described in Example 25. Strain NCFM Heat 1.1 was found to exhibit a higher effective productivity when stored for 7 d or 14 d than both control strains NCFM Wildtype C1 and NCFM Wildtype C2. However, only NCFM Heat 6.3 exhibited a higher effective productivity than the control culture NCFM Wildtype C2, but lower than NCFM Wildtype C1, when stored for 7 d or 14 d ( FIG. 29 ).

[0243] Example 27 Performance of strains NCFM Heat 1.1 and NCFM Heat 6.3 Seed cultures of NCFM Wildtype C1, NCFM Wildtype C2, NCFM Heat 1.1, and NCFM Heat 6.3 were generated following the three-step seed culture protocol described in the General Methods section. 7 mL of the second stage seed culture was inoculated into 343 mL of NCFM medium (Table 2). One vessel each was inoculated with NCFM Wildtype C1, NCFM Wildtype C2, NCFM Heat 1.1, and NCFM Heat 6.3. The cumulative base addition (Table 1) was recorded. The time course of cumulative base addition for the four fermentation runs is shown in FIG. 30.

[0244] NCFM Wildtype C1 was harvested after 10.98 hours, NCFM Wildtype C2 after 11.60 hours, NCFM Heat 1.1 after 12.20 hours, and NCFM Heat 6.3 after 12.80 hours. Final cell dry weight concentrations at harvest are shown in Figure 31. Cultures of NCFM Wildtype C1, NCFM Wildtype C2, NCFM Heat 1.1, and NCFM Heat 6.3 were harvested in 500 mL shake flasks, capped, and stored in a 10 C water bath for 1 hour. Cell concentrations determined after storage are shown in Figure 32.

[0245] Example 28 Performance of strains NCFM Heat 1.1 and NCFM Heat 6.3 in downstream processing The sample material generated in Example 27 was processed according to the protocol under "Downstream Processing in 96-well "Small-Scale" Plate Assay" described in the General Methods section. Along the processing steps, cell numbers were determined after the conditioning step as "CN" and a second time after downstream processing as "T0" as described in the General Methods section. Viability (downstream processing) was then calculated according to

number

[0246] Example 29 Storage stability of strains NCFM Heat 1.1 and NCFM Heat 6.3 Sample material produced in Example 28 was stored at 30° C., 40% relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 30° C., 40% relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 30° C., 40% relative humidity for 28 d (CFU (storage, 30° C., wet, 28 d)) using the method described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 30° C., wet, 28 d) was calculated according to the following:

number

[0247] Strain NCFM Heat 6.3 was found to have improved viability, i.e., improved storage stability, when stored at 30° C. and 40% relative humidity for 28 d compared to two control cultures, NCFM Wildtype C1 and NCFM Wildtype C1 (FIG. 34).

[0248] Sample material produced in Example 28 was stored at 30° C. at low relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 30° C. at low relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 30° C. at low relative humidity for 4 m (CFU (storage, 30C, dry, 4 m)) using the methods described for cell viability, plating, and statistical treatment in the General Methods section. The viability (storage, 30C, dry, 4 m) at each storage time X was calculated according to the following:

number

[0249] The results are shown in Figure 35.

[0250] Sample material produced in Example 28 was stored at 50° C. at low relative humidity according to the protocol described under "Storage stability testing by 96-well "small scale" plate assay at 50° C. at low relative humidity" in the General Methods section. The material was then tested for CFU content after storage at 50° C. at low relative humidity for 14 d (CFU (storage, 50° C., dry, 14 d)) using the method described for cell viability, plating, and statistical treatment in the General Methods section. Viability (storage, 50° C., dry, 14 d) was calculated according to the following:

number

[0251] The results are shown in Figure 36.

[0252] Example 30 Effective production capacity of NCFM Heat 1.1 Heat and NCFM Heat 6.3 under different storage conditions Information was obtained from culturing the strain as described in Example 27, processing the strain as described in Example 28, and analyzing the strain as described in Example 29 to derive the effective productivity of the strain according to the following:

number

[0253] In this example, for ΔCFU, we took the surviving "equivalent" cell concentration for the strain in the last stage of fermentation. The cells provided in the inoculum in this example were considered insignificant, and consequently the "equivalent" cell concentration for the strain was not corrected. fermentation For the sake of convenience, only the fermentation time for the strains in the last stage vessel was considered. After downstream processing and storage of the cultures for 14 d or 28 d at 30° C. and 40% relative humidity, strain NCFM Heat 6.3 was found to exhibit higher effective productivity than both control strains NCFM Wildtype C1 and NCFM Wildtype C2 ( FIG. 37 ).

[0254] Effective productivity determinations were performed. Strains were grown as described in Example 27, processed as described in Example 28, and stored at 30° C. and low relative humidity for 4 m as described in Example 29. The results are shown in FIG.

[0255] Effective productivity determinations were performed. Strains were grown as described in Example 27, processed as described in Example 28, and stored at 50° C. and low relative humidity for 14 d as described in Example 29. The results are shown in FIG.

[0256] Example 31 Genetic differentiation in thermally evolved Lactobacillus acidophilus NCFM Whole genome sequencing was performed on all selected clones essentially as described in General Methods. The genomes of two thermally evolved clones were sequenced and analyzed using breseq (Deatherage and Barrick 2014) as described in General Methods. The unevolved ancestor was also sequenced. Only mutations not present in the ancestor were considered. One of the clones, NCFM Heat 6.3, had nine mutations (Table 17), seven of which were shared by NCFM Heat 1.1. All mutations in NCFM Heat 1.1 (Table 18), except for one intergenic mutation, were in coding sequences. These likely affect gene function. One mutation in gntR resulted in a premature stop codon, which likely affected gene function. Two other mutations in the cation transporter and transposon were one nucleotide deletions, which likely caused a frameshift and affected gene function. The two additional mutations in NCFM Heat 6.3 were a synonymous mutation in rpsU and a non-synonymous substitution in threonyl-tRNA synthetase.

[0257] [Table 25]

[0258] [Table 26]

[0259] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. Brenner DM,Chey WD.2009.Bifidobacterium infantis 35624:a novel probiotic for the treatment of irritable bowel syndrome.Ann Rev Pharmacol Toxicol 9:7-15 Briczinski EP,Loquasto JR,Barrangou R,Dudley EG,Roberts AM,Roberts RF.2009.Strain-specific genotyping of Bifidobacterium animalis subsp.lactis by using single-nucleotide polymorphisms,insertions,and deletions.Appl Env Microbiol 75:7501-7508. Broadbent JR,Neeno-Eckwall EC,Stahl B,Tandee K,Cai H,Morovic W,Horvath P,Heidenreich J,Perna NT,Barrangou R,et al.2012.Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation.Genomics,12413,533. Brock TD.1989.Biotechnology:A Textbook of Industrial Microbiology.Sunderland,MA:Sinauer Associates,Inc. Castro HP,Teixeira PM,Kirby R.1997.Evidence of membrane damage in Lactobacillus bulgaricus following freeze drying.2017,Current Opinion Microbiology 82:87-94. Chouraqui JP,Van Egroo LD,Fichot MC.2004.Acidified milk formula supplemented with bifidobacterium lactis:impact on infant diarrhea in residential care settings.J Pediatr Gastroenterol Nutr 38:288-292. de Vrese M,Winkler P,Rautenberg P,Harder T,Noah C,Laue C,Ott S,Hampe J,Schreiber S,Heller K,et al.2005.Effect of Lactobacillus gasseri PA 16 / 8,Bifidobacterium longum SP 07 / 3,B.bifidum MF 20 / 5 on common cold episodes:a double blind,randomized,controlled trial.Clin Nutr 24:481-491. Deatherage DE,Traverse CC,Wolf LN,Barrick JE.2014.Detecting rare structural variation in evolving microbial populations from new sequence junctions using breseq.Front Genet 5:468. 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Claims

[Claim 1] A strain of Bifidobacterium animalis subsp. lactis deposited at the DSMZ under the number DSM 33461 on March 4, 2020; A strain of Lactobacillus acidophilus deposited at the DSMZ under number DSM 33463 on March 4, 2020; and A strain of Bifidobacterium animalis subsp. lactis, deposited at the DSMZ under the number DSM 33462 on March 4, 2020. A strain selected from

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