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WO2001012779A1 - Conservation de cellules bacteriennes a temperature ambiante - Google Patents

Conservation de cellules bacteriennes a temperature ambiante Download PDF

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Publication number
WO2001012779A1
WO2001012779A1 PCT/US2000/040704 US0040704W WO0112779A1 WO 2001012779 A1 WO2001012779 A1 WO 2001012779A1 US 0040704 W US0040704 W US 0040704W WO 0112779 A1 WO0112779 A1 WO 0112779A1
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drying
cells
fermentation
fermentation conditions
bacteria
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Victor Bronshtein
Charles Isaac
Gordana M. Djordjevic
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Universal Preservation Technologies Inc
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Universal Preservation Technologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • Patent No. 5,766,520 The foam-drying process addresses many of the drawbacks associated with freeze- or spray- drying and results in much lesser damage to numerous biological materials, including starter cultures.
  • bacteria are utilized in a wide range of commercial applications. Lactic acid bacteria cultures are used to produce cheese, yogurt, and other dairy products. Lactobacillus acidophilus, Bifidobacteria, E. coli and other types of bacteria are extensively used as probiotics. Live attenuated bacteria are extensively used to vaccinate different domestic animals and humans. Genetically altered bacteria are widely used as expression hosts for a variety of proteins and products. Unfortunately, broader applications of bacteria and other cell cultures are limited due to deficiencies in conventional preservation methods that do not allow effective stabilization and therefore distribution of cells at room and higher temperatures.
  • the present invention addresses various aspects of enhancing both the tolerance of bacterial cell cultures to desiccation by a scaleable foam-drying procedure and the survival of dried cells during subsequent storage at ambient temperatures.
  • the disclosed methods are based on the inventors' observations that certain modifications in bacterial cell culture conditions prior to foam-drying enhance the cells subsequent ability to survive desiccation and storage, possibly through the induction of intracellular protectant molecules.
  • a scaleable method for preserving bacterial cells in a dried state comprises culturing the bacterial cells under modified fermentation conditions that inhibit fermentation yield relative to optimal fermentation conditions and also enhance the cells' ability to survive dehydration, and drying the cell suspension by boiling under vacuum to form a mechanically-stable foam.
  • the modified fermentation conditions comprise fermentation parameters selected from the group consisting of temperature, pH, osmotic pressure, divalent cation concentration, cell density, nutrient concentration, oxygen concentration, and nitrogen concentration.
  • two or more of the fermentation parameters are modified to enhance the cells' ability to survive dehydration.
  • the modified fermentation conditions may be applied for only a portion of the culturing step.
  • the modified fermentation conditions may comprise at least increasing osmotic pressure of the media to 1.2- 10 times isotonic pressure.
  • the osmotic pressure may be increased by adding non-permeable solutes, permeable solutes, metabolized or non-metabolizable solutes.
  • the osmotic pressure is increased by adding at least one product of cell metabolism.
  • the modified fermentation conditions may comprise an increase or a decrease of about 0.5-4.5 pH units pH from an optimal pH.
  • the modified fermentation conditions may also comprise maintaining cultures to various non-optimal growth phases, such as early or late stationary phase, or early or late logarithmic phase.
  • the modified fermentation conditions may also comprise a change in temperature within a range of 1 -15° C from optimal.
  • Figure 1 shows stability of L. acidophilus harvested at either log or stationary phase.
  • Figure 2 shows stability of DSM L. acidophilus mixed with prebiotic and stored at room temperature.
  • Figure 3 shows a comparison of Bordetella survival following preservation by freeze-drying and by foam- drying.
  • bacterial cultures harvested in a stationary phase of growth survive desiccation better than cultures desiccated in a logarithmic phase. See for example, Legg U.S. Patent No. 5,728,574.
  • the use of nutrient starvation, heat shock and "osmoadaptation" by salt addition resulted in higher survival of
  • the present invention discloses effective preservation at ambient temperatures of bacterial cell cultures in industrial scale volumes by combining the both modification of conventional fermentation conditions, as suggested above, with the scaleable foam-drying preservation technology taught by Bronshtein, U.S. Pat. No. 5,766,520; incorporated herein in its entirety by reference thereto. Modification of fermentation was effective in enhancing preservation survival for a number of microorganisms, including: Lactococcus lactis subsp.
  • the methods and compositions of the present invention may encompass bacterial cells other than those specifically used in the below working examples. Further, other types of cell cultures are also deemed amenable to preservation using the disclosed techniques, such as for example, eubacteria, archaebacteria, protozoa, plankton (phyto- and zoo ), algae, fungi, mammalian (B-cells, fibroblasts, myoblasts, etc.) and insect cells.
  • the preserved cells may be used for attenuated bacteria or microorganizm-based vaccines, test kits and/or bioassays that require indicator cells, as well as any pre-packaged recombinant expression systems that use a defined cell line.
  • Foam-Drying Process In a preferred embodiment of the present invention, preservation is carried out by the foam-drying process detailed in U.S. Patent No. 5,766,520 to Bronshtein.
  • This so-called foam-drying protocol may include adding excipients, including carbohydrates and disaccharides to the biological suspension, and foaming the sample to thin films resulting in preservation by drying at ambient or higher temperatures.
  • the bacterial cells were generally mixed in preservation solutions prior to drying.
  • the suspensions were dried under vacuum and foamed to form thin films. Vitrification (glass formation) may or may not occur depending on the drying and storage conditions as detailed by Bronshtein, U.S. Pat. No. 5,766,520.
  • the dried samples were stored under vacuum.
  • polyols and polymers are known in the art and may serve as protectants as long as they enhance the ability of the cells to withstand drying and storage. Indeed, the protectant molecules provide other advantages during preservation (see infra, as an aid to generating mechanically stable foams) besides stabilizing cells during dehydration.
  • the protectants in accordance with the present invention may include, without limitation, simple sugars, such as sucrose, glucose, maltose, sucrose, xylulose, ribose, mannose, fructose, raffinose, and trehalose, non-reducing derivatives of monosaccharides and other carbohydrate derivatives, sugar alcohols like sorbitol, synthetic polymers, such as polyethylene glycol, hydroxyethyl starch, polyvinyl pyrrolidone, polyacrylamide, and polyethyleneamine, and sugar copolymers, like Ficoll and Dextran, and combinations thereof. Low molecular weight proteins that are soluble in the cell suspension may also serve as protectants.
  • simple sugars such as sucrose, glucose, maltose, sucrose, xylulose, ribose, mannose, fructose, raffinose, and trehalose
  • non-reducing derivatives of monosaccharides and other carbohydrate derivatives sugar alcohol
  • the protective composition may comprise mixtures of a low molecular weight sugar, a disaccharide, oligosaccharide and polymer, including a biological polymer.
  • the low molecular weight sugar is used to penetrate and protect intracellular structures during dehydration.
  • Low molecular weight, permeating sugars may be selected from a variety of ketoses, which are non-reducing at neutral or higher pH, or methylated or ethylated monosaccharides.
  • non-reducing ketoses are included: the six carbon sugars, fructose, sorbose, and piscose; the five carbon sugars, ribulose and xylulose; the four-carbon sugar, er ⁇ thulose; and the three-carbon sugar, 1,3 dihydroxydimethylketone.
  • methylated monosaccharides are the alpha and beta methylated forms of gluco, ma ⁇ no, and galacto p ⁇ ranoside.
  • methylated five carbon compounds are the alpha and beta forms of arabino and xylo pyranosides.
  • Disaccharides like sucrose, are known to be effective protectants during desiccation because they replace the water of hydration on the surface of biological membranes and macromolecules.
  • sucrose and/or other fillers may be effectively transformed into a stable foam composed of thin amorphous films of the concentrated sugar when dried under vacuum.
  • a polymer may be employed to increase the glass transition temperature (Tg) of the dehydrated mixture, which may be decreased by inclusion of the low molecular weight monosaccharides.
  • Any biological polymers that are highly soluble in concentrated sugar solutions may be employed.
  • polysaccharides, like Ficoll, and Dextran, and synthetic polymers, like hydroxyethyl starch, polyethylene glycol, polyvinyl pyrrolidone, polyacrylamide, as well as highly soluble natural and synthetic biopolymers (e.g. proteins) will help to stabilize biological membranes and increase Tg.
  • desiccation of the bacterial cell cultures is preferably accomplished by foam-drying to form a mechanically stable porous structure by boiling under a vacuum.
  • the drying step may be carried out at temperatures in the range of about -15 to 70 C.
  • the mechanically stable porous structure, or foam consists of thin amorphous films of the concentrated fillers.
  • Preservation by foam formation is particularly well suited for efficient drying of large sample volumes, before vitrification, and as an aid in preparing a readily milled dried product suitable for commercial use.
  • dilute culture suspensions may be concentrated by partially removing the water to form a viscous specimen before foam-drying under vacuum.
  • This initial concentration step can be accomplished either before or after introduction of the sample into the processing chamber, depending on the concentration method chosen. Alternatively, some samples may be sufficiently viscous after addition of the protectant molecules, and therefore not require any initial concentration. In situations where it is desirable to increase the viscosity of the samples, methods contemplated for use in initial concentration include freeze-drying, evaporation from liquid or partially frozen state, reverse osmosis, other membrane technologies, or any other concentration methods known in the art.
  • the samples are subjected to vacuum, to cause them to boil during drying at temperatures substantially lower than 100 C.
  • reduced pressure is applied to solutions or suspensions of biologically active materials to cause the solutions or suspensions to foam during boiling, and during the foaming process further solvent removal causes the ultimate production of a mechanically-stable open-cell or closed-cell porous foam.
  • low vacuum pressures in the range of 0.1-0.9 atm
  • much higher vacuum pressures (0-24 Torr) are used to cause boiling.
  • the vacuum for the boiling step is preferably 0-10 Torr, and most preferably less than about 4 Torr. Boiling in this context means nucleation and growth of bubbles containing water vapor, not air or other gases. In fact, in some solutions, it may be advantageous to purge dissolved gases by application of low vacuum (about 0.1-0.9 atm) at room temperature.
  • “degassing” may help to prevent the solution from erupting out of the drying vessel. Once the solution is sufficiently concentrated and viscous, high vacuum can be applied to cause controlled boiling or foaming. Concentration of the protectant molecules recited above, in the range of 5-70% by weight, during initial evaporation aids in preventing freezing under subsequent high vacuum and adds to the viscosity, thereby facilitating foaming while limiting uncontrolled eruptions. Rapid increases in pressure or temperature could cause a foam to collapse. In this case, to enhance the mechanical stability of the porous structures, surfactants may be added as long as those additives do not interfere with the biological activity of the solute intended for conversion to dry form. Moreover, drying of the protectant polymers also contributes to the mechanical stability of the porous structures.
  • Foams prepared according to the present invention may be stored in the processing chamber under vacuum, dry gas, like N 2 atmosphere and/or chemical desiccant, prior to subsequent processing operations, (e.g. stability drying, vitrification or milling).
  • dry gas like N 2 atmosphere and/or chemical desiccant
  • subsequent processing operations e.g. stability drying, vitrification or milling.
  • the following examples illustrate the foam-drying process as applied to bacterial cell cultures that have not been subjected to modification of fermentation conditions prior to drying:
  • the frozen suspension (6 g) was thawed at 4 C and mixed with 4 g of 9:1 sucrose: maltrin mixture. The sample was mixed until the sugars were completely dissolved, so that the final suspension contained 35 wt% sucrose and 4 wt% maltrin.
  • the suspension was placed inside 20 ml vials at 2 g per vial. The vials were dried inside a vacuum chamber. The vials were sitting on the surface of stainless steel shelf inside the chamber. The shelf temperature was controlled by circulating ethylene glycol/water antifreeze at a controlled temperature inside the shelf. Before the vacuum was applied the shelf temperature was decreased to 5 C. The hydrostatic pressure inside the chamber was then decreased to 0.5 Torr. Under such conditions, the suspension boiled for 30 min. The temperature of the shelf was then slowly (during 30 min) increased up to
  • Ice nucleating activity of preserved INB was measured after the samples were rehydrated with 10 ml of 0.01 M phosphate buffer. Ice nucleating activity was measured as a concentration of ice nucleating centers that nucleate an ice crystal in a 10 ul buffer drop during 5 min at -5 C.
  • the ice nucleating activity of the samples that had been removed from the vacuum chamber after drying at 25 C was approximately 50% less than the initial activity of frozen-thawed INB. (The relative standard error in the measurement of ice nucleating activity is less than 20%). Because, it is known that freezing of INB does not significantly decrease ice nucleating activity, the 50% decrease of the activity observed in this experiment is probably because the additional freezing step increases sensitivity of INB to preservation by drying. At the same time, no additional decrease of the activity of the INB was observed after an additional 7 days drying at 50 C under vacuum. (4) When stable foams containing INB, prepared as above, were subjected to milling using a modified Virtis homogenizer, there was no loss of ice nucleating activity in the rehydrated powder, compared to the rehydrated foam.
  • the culture medium (5 ml) was pipetted into a centrifuge tube and centrifuged for 10 min. The supernatant was then poured off and the weight of the pellets was measured to determine the approximate concentration of the cells.
  • the bacterial cells were resuspended with 5 ml of NZYM broth or preservation solution consisting of 25% sucrose and 25% fructose in MRS broth. The cells resuspended with NZYM broth were used as a control.
  • the cells suspended in 25% sucrose and 25% fructose in MRS broth (1 mi) were placed in 20 ml glass vials and dried under vacuum similar to the INB were dried in the Example #1. After that, the samples were kept under vacuum up to 24 days at room temperature. Dried samples were assayed at selected time intervals. The survival of the preserved cells was measured after reh ⁇ dration with 0.1 % peptone solution in water at room temperature. To determine concentration of viable cells the suspensions were pour plated in Petri dishes at the appropriate dilution on LB Miller agar followed by incubation at 37 C for 36-48 hours. Approximately 25 ⁇ 10% of control cells survived after drying and one day of storage under vacuum.
  • Optional Stability-Drying The mechanically stable foams formed during primary foam-drying, may optionally undergo secondary or "stability" drying at increased temperatures. Since Tg is dependent on the water content of the sample and since Tg increases with increased dehydration, different stability-drying protocols may be applied depending on the desired storage temperature, to generate a Tg consistent with vitrification upon cooling to that storage temperature. However, because dehydration of materials is practically impossible once they have entered the glass state, the key to vitrification according to the present invention, where ambient storage temperatures may be desired, is to conduct the stability drying at a temperature significantly higher than the ambient temperature.
  • Ultimate storage temperatures are preferably within the range of 0 -70 C. More preferably, common storage temperature selections are greater than or equal to 0 , 4 , 20 , 40 , and 50 C. In some cases, where refrigerated storage may be preferred, stability-drying could be carried out at room temperature followed by cooling to the storage temperature or below. In other instances, however, where stability at room temperature is desired, dehydration at a temperature above room temperature should be employed, followed by cooling to room temperature.
  • the nature and stability characteristics of the cells will determine the maximum temperature they can withstand during the foam-drying and stability-drying steps.
  • the stability-drying temperature may be increased to higher temperatures without loss of viability.
  • Selection and optimization of foam-drying and stability-drying parameters is preferably performed for each strain of bacteria to be preserved, using small sample volumes. A range of foam-drying temperatures and pressures, which are together sufficient to effect boiling of the suspension, may be tested. Once a mechanically-stable foam has formed, cell viability can be measured to determine optimal foam-drying conditions.
  • the temperatures and pressures to which foam-dried cultures are exposed may be varied to provide a range of stability- drying parameters.
  • cell viability serves as an indicator of optimal stability-drying conditions.
  • continuous or step-wise increases in the stability-drying temperature may be used to place labile cells in a state of thermal stability at storage temperatures that may be lethal for native cultures.
  • the stability-drying temperature is above a desired storage temperature. In addition to conducting the stability-drying at temperatures above the selected storage temperature, it is preferred that this drying is carried out for a period of time sufficient to actually raise Tg above the storage temperature.
  • Tg is actually greater than the storage temperature
  • DSC Differential scanning calorimetry
  • TSPC Thermally Stimulated Polarization Current
  • the dried foams may be stored at a selected storage temperature for a selected storage period.
  • dried bacterial cultures can be rehydrated with culture medium and assayed for viability.
  • viable cell counts are determined by serial dilution of the sample and plating on appropriate agar. The plates are incubated and colony-forming units (CFU) determined. Percent survival can be calculated by dividing the surviving CFU/ml of the sample being tested by the CFU/ml of the original fermentation.
  • the fermentation process can be defined as a chemical transformation of organic compounds by the metabolic activity of the microorganisms.
  • Numerous commercially important products are made by fermentation, including microbial cells (starter cultures), large macromolecules (enzymes, gums, etc.), primary metabolic end products (lactic acid, flavor compounds, etc.), and secondary metabolites (antibiotics, etc.).
  • microbial fermentation is considered a multi dimensional process, comprising many parameters including; culture temperature, pH, osmotic strength, time of cell growth (cell density), ionic strength, concentration of bivalent cations, and media composition (e.g., nutrient, oxygen, nitrogen concentration, etc.).
  • media composition e.g., nutrient, oxygen, nitrogen concentration, etc.
  • Optimal fermentation conditions are those which result in maximal bacterial growth, metabolic activity and cell density (fermentation yield). However, these optimal fermentation conditions usually do not result in a culture containing cells that exhibit optimal desiccation tolerance.
  • Our approach is to find the combination of fermentation parameters that would yield maximal cell survival after fermentation, preservation by foam-drying (with or without optional stability-drying), and subsequent storage at temperatures required for the practical application of the particular cell strain.
  • Bacteria have developed numerous mechanisms to cope with non-optimal growth environments. The exposure of cells to different stresses is known to have an immediate impact on bacterial physiology. We have found that the growth of cellular (bacterial) cultures under certain sub-optimal fermentation conditions increases the desiccation tolerance of many bacterial strains. In contrast the previous work of Legg (U.S. Pat. No. 5,728,574), a more effective means of enhancing desiccation tolerance may be found by the simultaneous modification of all or several different fermentation parameters.
  • Temperature Most bacterial species are able to grow over a wide range of temperatures, up to 40° C. The effect of temperature on bacterial growth has kinetics similar to that on the rate of a chemical reaction and which is described by the so called “Arrhenius plot" (curve has hyperbolic shape).
  • the optimal, maximal, and minimal growth temperatures are called “cardinal temperatures”.
  • the optimal temperature is defined as the temperature at which bacterial growth occurs at a maximal rate.
  • the growth temperature range between the maximum and optimal temperature is called the “high growth range”.
  • the growth range between the minimum and the optimal growth temperature is called the "low growth range”.
  • Bacterial growth is linear in the optimal temperature range. The slope of the growth curve increases in the low and high growth range, and becomes vertical at inhibitory (lethal) temperatures.
  • the nutritive characteristics of the growth medium can affect growth at the low and high temperature ranges, but have no effect on the optimal temperature of growth. Enriching the growth medium is known to have an effect on the maximum growth temperature of E. coli, but not on the minimum growth temperature. Based on temperature ranges where growth occurs, bacteria are classified as “psychrophiles” (can grow at +5° C or below, and up to 40° C), “mesophiles” (growth range: from + 5° C to 45-50° C, optimal growth at 37° C; examples E. coli, Lactobacilli, etc.), and “thermophiles” (growth range from 40° C to above 100° C).
  • bacteria activate their "heat shock” stress responses, change composition of the phospholipids in biological membranes, etc.
  • the maximum growth temperature of many bacteria is determined by the thermal instability of their proteins.
  • minimum growth temperature is set by factors that cause weakening of h ⁇ drophobic bonds involved in higher levels of protein structural organization.
  • Bacteria can grow over a wide ranges of pH's. However, bacteria maintain their internal pH near a fixed optimal rate, which is defined by the optimal pH for catalytic activity of the enzymes necessary for bacterial growth. Depending of the pH of the growth medium, the bacterial cytoplasm could be either more acidic or more alkaline than the medium. For example. E. coli can grow over pH ranges of 6.0 to 8.0, but always maintains an internal pH at 7.6. The Lactobacillus species are known to tolerate external pH as low as 3.5, but these bacteria maintain the internal pH of 7.6. However, because they have extremely efficient proton pumps, Lactobacilli can survive internal pH as low as 4.4. Efficient pumping-out of protons from the cytoplasm seems to be the general strategy that bacteria developed to cope with a low pH.
  • Osmotic pressure All bacteria, with the exception of mycoplasmas, have developed versatile strategies to maintain a characteristic turgor pressure over a relatively broad range of osmotic strengths of their external environment. Generally, Gram-positive bacteria maintain higher turgor (5-22 atm) than Gram-negative bacteria (0.8-5 atm). Turgor pressure is maintained mostly by adjusting the intracellular concentration of so called “compatible solutes", small, neutral organic molecules, which are highly soluble and do not alter cytoplasmic functions. Compatible solutes can be accumulated by either de novo synthesis or by transport into the cells after osmotic shock.
  • solutes including betaine (N, N, N-trimethylglycine), carnitine, trehalose, sucrose, glucitol, ectoine, mannitol, proline, glycerol, small peptides, etc. (reviewed by Csonka. 1991 Annu. Rev. Microbiol. 45:569-606).
  • Some of the listed compounds are also widely known as "fillers” (i.e., trehalose, sucrose, manitol, glycerol, and glucitol).
  • HSLs homoserine lactones
  • small peptides mediate cell-cell communication in Gram-positive bacteria.
  • Signaling molecules so called “pheromones” or “autoinducers”, are produced and secreted into the growth medium at a basal level at low cell densities. The concentration of the pheromones increases with cell density until a threshold level is reached, and could be recovered from the supernatant after centrifugation of the culture. Pheromones usually enter the cells via diffusion or by a dedicated transport system.
  • signaling molecules interact with different effector molecules directly or via a two-component sensing systems consisted of His-Kinase and a response regulator protein.
  • Cell-cell communication was described in several bacterial genera, including Vibrio, Streptococcus, Enterococcus, Pseudomonas, Myxococcus, Bacillus, Agrobacterium, Erwinia, Rhizobium, Xanthomo ⁇ as, Staphylococcus, Lactococcus, Lactobacillus, and Streptomyces.
  • Divalent Cations The composition of the fermentation broth will include a large number of divalent cations.
  • the fermenter was equipped with a pH module to control pH by the addition of acid or base to the fermenting culture as necessary.
  • the starting shelf temperature was set to 7° C.
  • Bacterial cells were suspended in a concentrated sucrose solution and aliquoted into serum vials.
  • a thermocouple was placed into one of the samples to monitor sample temperature during the preservation process.
  • the vacuum chamber was closed and the pressure reduced to 5 Torr.
  • sample temperature had dropped to about 0° C, due to evaporational cooling, the shelf temperature was increased to 20° C.
  • the pressure was dropped incrementally such that the sample temperature never fell below -10° C.
  • the final pressure was 200 mTorr.
  • the shelf temperature was increased to 25° C for stability-drying. After 15 hours at 25° C, the shelf temperature was increased again to 45° C.
  • the samples remained under vacuum at 45° C for 36 hours.
  • the samples were sealed under vacuum prior to being removed from the vacuum chamber.
  • CFU colony-forming units
  • Example 1 Fermentation of Lactobacillus acidophilus (ATCC 4356) at Low pH Lactobacillus acidophilus ATCC 4356 (L. acidophilus) is a commercially significant species.
  • L. acidophilus grows by fermentation of lactose, glucose and a range of carbohydrates. The end product of this fermentation is almost exclusively lactic acid. If the lactic acid produced is not neutralized by the addition of base, the pH of the culture decreases. L. acidophilus, and other lactic acid bacteria will produce acid to the point their growth is curtailed by the low pH.
  • L. acidophilus ATCC 4356 was also fermented in a modified manner by allowing the pH of the culture to fall with no regulation.
  • the cells were fermented to stationary phase indicated by an O.D. of 2.4.
  • the conditions were identical to the conventional fermentation but the pH was allowed to drop to the point it restricted growth.
  • the survival of these cells (no pH regulation) following foam-drying and rehydration was over 70%.
  • L. acidophilus fermented with no pH regulation to an O.D. ((5 ) 600 nm) greater than 2.4 and a final pH of lower than 4.0 was dried with a 70% level of survival after re hydration. Intermediate levels of survival after foam-drying and rehydration, between 20% and 70%, were observed when L. acidophilus was grown under conditions in which the cultures did not completely achieve and O.D. of greater than 2.4 and a pH lower than 4.0. If the O.D. exceeded 2.4, then the pH was lower than 4.0, and vice-versa. The enhanced desiccation tolerance was observed when the samples were foam-dried in both 20 uL drops and in foams of larger volumes.
  • the first two time points show cell counts prior to foam-drying and following the drying process, respectively.
  • the cell counts remained essentially stable throughout the 17 day storage period at 37° C.
  • the cell density was 4.2x10 8 CFU/mL in the log phase versus 1.0x10 9 CFU/mL in the stationary phase.
  • Example 4 Effect of Depleted Supernatant on Desiccation Tolerance of L. acidophilus
  • Depleted supernatant was prepared by inoculating a 200 mL stir flask containing MRS + 0.05% cysteine with frozen seed. This culture was incubated at 37° C with no pH regulation until the optical density (at 600 nm) was greater than 2.60 and the pH of the culture was less than 4.0. The culture was removed from the stir and centrifuged. The supernatant was decanted off the pellet. The collected supernatant was filtered through a 0.22 urn filter. Samples of the depleted supernatant were neutralized by the addition of 1 M NaOH until the pH reached 7.0.
  • Bacteria were enumerated by plating on MRS + 0.05% cysteine agar and incubating for 48 h at 37° C under anaerobic conditions.
  • the treatment with depleted supernatant increased the preservation survival of log phase cells from 5-10% to 20-30%.
  • the observed increase in preservation survival after treatment with depleted supernatant was less in stationary phase cells compared to log phase cells fermented under pH regulation.
  • the effect of the depleted supernatant was observed with neutralized as well as native depleted supernatant
  • Example 5 Desiccation Tolerance of L. acidophilus DSM strain Bacteria were fermented in Difco MRS + 0.05% cysteine broth with the addition of 0.1 % Ca +2 at a temperature of 42° C. The pH was regulated at 5.80. Cells were harvested in stationary phase as determined by a stabilization of optical density. The bacteria were preserved by foam-drying with a yield of 55%. Bacteria were enumerated by plating on MRS + 0.05% cysteine agar and incubating for 48 h at 37° C under anaerobic conditions.
  • the preserved bacteria were prepared as described.
  • the mechanically-stable foam glass was milled to a fine powder.
  • the milled glass containing the preserved bacteria was mixed with an inulin powder under dry conditions (r.h. ⁇ 20%).
  • the mixture was sealed in foil pouches and stored at room temperature.
  • the bacteria were preserved with a yield of 62%.
  • the bacteria in the mixed powder were stable for 86 days at 25° C.
  • Example 6 Growth to Stationary Phase of Salmonella choleraesuis S. choleraesuis was fermented by conventional methods in M-broth comprising Difco M-broth (36 g), Tris 7-9 buffer salt (12.0 g), 10% Phenol red (2.0 mL), deionized water to 1000 mL and adjusted to pH 7.4. Fermentation pH was adjusted to 7.1 by the addition of 1 HCI or 1 N NaOH. Stationary phase was defined as the stabilization of optical density at 650 nm. The cells were harvested and concentrated by centrifugatio ⁇ before preservation. The bacteria were preserved by foam-drying. Bacteria were enumerated by plating on Difco Trypticase Soy Agar (TSA) and incubating for 24 hours at 37° C.
  • TSA Difco Trypticase Soy Agar
  • Dextrose Starch Broth is composed of dextrose (2.0 g), soluble starch (10.0 g), NaCI (5.0 g), disodium phosphate (3.0 g), gelatin (bacteriological) (20.0 g), glycerol (10.0 g), sodium acetate (0.08 g), deionized water to 1000 mL and pH adjusted to 7.3.
  • the culture was grown to late stationary phase as defined by the stabilization of O.D.
  • the bacteria were preserved by foam-drying. The bacteria were enumerated by plating on Bordet Gengou + 5% blood agar and incubating at 37° C under aerobic conditions.
  • Lactococcus lactis subsp. cremoris ATCC 19257 was fermented in skim milk-based medium at 30° C. The pH of the culture was maintained at 5.80 by the addition of 4.76% NH 4 0H. The cells were harvested in stationary phase as defined by the halt in addition of base to the culture. The bacteria were preserved by foam-drying. The bacteria were enumerated by plating on TSA and incubating at 30° C for 48 hours. After drying the cells fermented under these conditions, survival was less than 18%.
  • the conventional fermentation described above was modified by the addition of a non-metabolized sugar (sucrose) to the fermentation broth.
  • Sucrose was added at a concentration of 20%.
  • Lactococcus lactis subsp. cremoris ATCC 19257 was fermented as above in a broth of MRS + C broth +20% Sucrose. The preservation survival after drying was 100%.

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  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne l'amélioration de la survie de cellules bactériennes au cours du séchage et du stockage de celles-ci, par une modification de leurs conditions de culture avant déshydratation pour former une mousse. Les conditions de culture modifiées peuvent entraîner la production d'agents protecteurs.
PCT/US2000/040704 1999-08-19 2000-08-21 Conservation de cellules bacteriennes a temperature ambiante Ceased WO2001012779A1 (fr)

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CA002382061A CA2382061A1 (fr) 1999-08-19 2000-08-21 Conservation de cellules bacteriennes a temperature ambiante
EP00969011A EP1402003A4 (fr) 1999-08-19 2000-08-21 Conservation de cellules bacteriennes a temperature ambiante

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US14979599P 1999-08-19 1999-08-19
US60/149,795 1999-08-19

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010064222A2 (fr) 2008-12-01 2010-06-10 University College Cork, National University Of Ireland, Cork Igf1 pour réparation myocardique
US20110189654A1 (en) * 2008-05-27 2011-08-04 Qiagen Gmbh Diagnostic reagent, containing bioparticles, method for production thereof and use thereof as internal standard in nucleic acid preparation and nucleic acid detection methods
US8097245B2 (en) 2005-12-28 2012-01-17 Advanced Bionutrition Corporation Delivery vehicle for probiotic bacteria comprising a dry matrix of polysaccharides, saccharides and polyols in a glass form and methods of making same
US8968721B2 (en) 2005-12-28 2015-03-03 Advanced Bionutrition Corporation Delivery vehicle for probiotic bacteria comprising a dry matrix of polysaccharides, saccharides and polyols in a glass form and methods of making same
US9072310B2 (en) 2006-12-18 2015-07-07 Advanced Bionutrition Corporation Dry food product containing live probiotic
US9504275B2 (en) 2010-08-13 2016-11-29 Advanced Bionutrition Corporation Dry storage stabilizing composition for biological materials
US9504750B2 (en) 2010-01-28 2016-11-29 Advanced Bionutrition Corporation Stabilizing composition for biological materials
US9623094B2 (en) 2009-03-27 2017-04-18 Advanced Bionutrition Corporation Microparticulated vaccines for the oral or nasal vaccination and boostering of animals including fish
EP3167845A1 (fr) 2015-11-12 2017-05-17 The Provost, Fellows, Foundation Scholars, & the other members of Board, of the College of Holy and Undiv. Trinity of Queen Elizabeth near Dublin Extenseur biocompatible implantable permettant de traiter des étranglements de lumière corporelle
US9731020B2 (en) 2010-01-28 2017-08-15 Advanced Bionutrition Corp. Dry glassy composition comprising a bioactive material
CN108823099A (zh) * 2018-07-16 2018-11-16 广州富诺营养科技有限公司 一种固态益生菌制剂的制备方法及其制剂
US10293006B2 (en) 2016-01-07 2019-05-21 Ascus Biosciences, Inc. Microbial compositions for improving milk production in ruminants
US10844419B2 (en) 2015-06-25 2020-11-24 Native Microbials, Inc. Methods, apparatuses, and systems for analyzing microorganism strains from complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
US10851399B2 (en) 2015-06-25 2020-12-01 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
US10953050B2 (en) 2015-07-29 2021-03-23 Advanced Bionutrition Corp. Stable dry probiotic compositions for special dietary uses
US11044924B2 (en) 2017-04-28 2021-06-29 Native Microbials, Inc. Methods for supporting grain intensive and or energy intensive diets in ruminants by administration of a synthetic bioensemble of microbes or purified strains therefor
US11214597B2 (en) 2009-05-26 2022-01-04 Advanced Bionutrition Corp. Stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making
US11891647B2 (en) 2016-12-28 2024-02-06 Native Microbials, Inc. Methods, apparatuses, and systems for analyzing complete microorganism strains in complex heterogeneous communities, determining functional relationships and interactions thereof, and identifying and synthesizing bioreactive modificators based thereon
US12018313B2 (en) 2016-12-28 2024-06-25 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities with tracer analytics, determination of functional relationships and interactions thereof, and synthesis of microbial ensembles

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998024882A1 (fr) * 1996-12-05 1998-06-11 Quadrant Holdings Cambridge Limited Methodes de conservation de cellules procaryotes et compositions resultantes
US5766520A (en) * 1996-07-15 1998-06-16 Universal Preservation Technologies, Inc. Preservation by foam formation

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4797365A (en) * 1985-01-22 1989-01-10 Nabisco Brands, Inc. Active dried yeast
GB9314886D0 (en) * 1993-07-19 1993-09-01 Zeneca Ltd Production of a biological control agent
CZ391297A3 (cs) * 1995-06-07 1998-05-13 Quadrant Holdings Cambridge Limited Způsob přípravy tenké porovité skelné matrice a prostředek tím získaný

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5766520A (en) * 1996-07-15 1998-06-16 Universal Preservation Technologies, Inc. Preservation by foam formation
WO1998024882A1 (fr) * 1996-12-05 1998-06-11 Quadrant Holdings Cambridge Limited Methodes de conservation de cellules procaryotes et compositions resultantes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LOEWEN ET AL.: "Regulation in the RpoS regulon of escherichia coli", CANADIAN JOURNAL OF MICROBIOLOGY, vol. 44, no. 8, August 1998 (1998-08-01), pages 707 - 717, XP002936640 *
NOTLEY ET AL.: "Induction of RpoS-dependent functions in glucose-limited continuous culture: What level of nutrient limitation induces the stationary phase of escherichia coli?", JOURNAL OF BACTERIOLOGY, vol. 178, no. 5, March 1996 (1996-03-01), pages 1465 - 1468, XP002936639 *
See also references of EP1402003A4 *
STROM ET AL.: "Trehalose metabolism in escherichia coli: Stress protection and stress regulation of gene expression", MOLECULAR MICROBIOLOGY, vol. 8, no. 2, 1993, pages 205 - 210, XP002936641 *

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US9737578B2 (en) 2005-12-28 2017-08-22 Advanced Bionutrition Corp. Delivery vehicle for probiotic bacteria comprising a dry matrix of polysaccharides, saccharides and polyols in a glass form and methods of making same
US8097245B2 (en) 2005-12-28 2012-01-17 Advanced Bionutrition Corporation Delivery vehicle for probiotic bacteria comprising a dry matrix of polysaccharides, saccharides and polyols in a glass form and methods of making same
US8968721B2 (en) 2005-12-28 2015-03-03 Advanced Bionutrition Corporation Delivery vehicle for probiotic bacteria comprising a dry matrix of polysaccharides, saccharides and polyols in a glass form and methods of making same
US9044497B2 (en) 2005-12-28 2015-06-02 Advanced Bionutrition Corporation Delivery vehicle for probiotic bacteria comprising a dry matrix of polysaccharides, saccharides and polyols in a glass form and methods of making same
US9072310B2 (en) 2006-12-18 2015-07-07 Advanced Bionutrition Corporation Dry food product containing live probiotic
US9480276B2 (en) 2006-12-18 2016-11-01 Advanced Bionutrition Corporation Dry food product containing live probiotic
US20110189654A1 (en) * 2008-05-27 2011-08-04 Qiagen Gmbh Diagnostic reagent, containing bioparticles, method for production thereof and use thereof as internal standard in nucleic acid preparation and nucleic acid detection methods
EP3047861A2 (fr) 2008-12-01 2016-07-27 University College Cork-National University of Ireland, Cork Igf1 pour réparation du myocarde
WO2010064222A2 (fr) 2008-12-01 2010-06-10 University College Cork, National University Of Ireland, Cork Igf1 pour réparation myocardique
US9623094B2 (en) 2009-03-27 2017-04-18 Advanced Bionutrition Corporation Microparticulated vaccines for the oral or nasal vaccination and boostering of animals including fish
US11214597B2 (en) 2009-05-26 2022-01-04 Advanced Bionutrition Corp. Stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making
US10575545B2 (en) 2010-01-28 2020-03-03 Advanced Bionutrition Corp. Stabilizing composition for biological materials
US9731020B2 (en) 2010-01-28 2017-08-15 Advanced Bionutrition Corp. Dry glassy composition comprising a bioactive material
US9504750B2 (en) 2010-01-28 2016-11-29 Advanced Bionutrition Corporation Stabilizing composition for biological materials
US10206421B2 (en) 2010-01-28 2019-02-19 Advanced Bionutrition Corp. Stabilizing composition for biological materials
US9504275B2 (en) 2010-08-13 2016-11-29 Advanced Bionutrition Corporation Dry storage stabilizing composition for biological materials
US10851399B2 (en) 2015-06-25 2020-12-01 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
US10844419B2 (en) 2015-06-25 2020-11-24 Native Microbials, Inc. Methods, apparatuses, and systems for analyzing microorganism strains from complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
US10953050B2 (en) 2015-07-29 2021-03-23 Advanced Bionutrition Corp. Stable dry probiotic compositions for special dietary uses
EP3167845A1 (fr) 2015-11-12 2017-05-17 The Provost, Fellows, Foundation Scholars, & the other members of Board, of the College of Holy and Undiv. Trinity of Queen Elizabeth near Dublin Extenseur biocompatible implantable permettant de traiter des étranglements de lumière corporelle
WO2017081326A2 (fr) 2015-11-12 2017-05-18 The Provost, Fellows, Fdn Scholars, & The Other Members Of Board, Of The College Of The Holy & Undiv. Trinity Of Queen Elizabeth Détendeur biocompatible implantable approprié pour le traitement de constrictions de lumière corporelle
US10398154B2 (en) 2016-01-07 2019-09-03 Ascus Biosciences, Inc. Microbial compositions and methods of use for improving milk production
US11910809B2 (en) 2016-01-07 2024-02-27 Native Microbials, Inc. Microbial compositions and methods of use for improving milk production
US10701955B2 (en) 2016-01-07 2020-07-07 Ascus Biosciences, Inc. Ruminant compositions
US10448657B2 (en) 2016-01-07 2019-10-22 Ascus Biosciences, Inc. Cow food and methods of husbandry for increased milk production
US10448658B2 (en) 2016-01-07 2019-10-22 Ascus Biosciences, Inc. Cow food and methods of husbandry for increased milk production
US10293006B2 (en) 2016-01-07 2019-05-21 Ascus Biosciences, Inc. Microbial compositions for improving milk production in ruminants
US10966437B2 (en) 2016-01-07 2021-04-06 Native Microbials, Inc. Microbial compositions and methods of use for improving milk production
US10645952B2 (en) 2016-01-07 2020-05-12 Ascus Biosciences, Inc. Microbial compositions and methods of use for improving milk production
US11291219B2 (en) 2016-01-07 2022-04-05 Native Microbials, Inc. Microbial compositions and methods of use for improving milk production
US11910808B2 (en) 2016-01-07 2024-02-27 Native Microbials, Inc. Ruminant compositions
US12018313B2 (en) 2016-12-28 2024-06-25 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities with tracer analytics, determination of functional relationships and interactions thereof, and synthesis of microbial ensembles
US11891647B2 (en) 2016-12-28 2024-02-06 Native Microbials, Inc. Methods, apparatuses, and systems for analyzing complete microorganism strains in complex heterogeneous communities, determining functional relationships and interactions thereof, and identifying and synthesizing bioreactive modificators based thereon
US11044924B2 (en) 2017-04-28 2021-06-29 Native Microbials, Inc. Methods for supporting grain intensive and or energy intensive diets in ruminants by administration of a synthetic bioensemble of microbes or purified strains therefor
US11871767B2 (en) 2017-04-28 2024-01-16 Native Microbials, Inc. Microbial compositions and methods for ruminant health and performance
CN108823099A (zh) * 2018-07-16 2018-11-16 广州富诺营养科技有限公司 一种固态益生菌制剂的制备方法及其制剂

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EP1402003A1 (fr) 2004-03-31
CA2382061A1 (fr) 2001-02-22
EP1402003A4 (fr) 2004-07-14

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