Note: Descriptions are shown in the official language in which they were submitted.
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PRESERVATION OF BACTERIAL CELLS AT AMBIENT TEMPERATURES
Back4round of the Invention
Preservation by drying has been known for thousands of years. Modern
refinements of this art are evident
in freezing, freeze-drying, drying from liquid, and desiccation. The key step
is to remove the water that allows the
harmful interactions to occur. The art has been successfully carried out on
many biological materials including
microorganisms. However this development has been variable in both type of
organisms that can be successfully
preserved and variability with preservation of that organism.
Conventional preservation methods, including freeze-drying and spray drying,
provide only limited success in
delivery of metabolically active cells present at high densities. Cell
injuries inherently associated with these methods
of preservation result in starter cultures with low metabolic activity and
cell density, causing long "lag" phases in
commercial fermentations and economic losses. Recently, Bronshtein has
developed an alternative foam-drying
process for the long-term stabilization of sensitive biological materials at
ambient temperatures in the dry state (U.S.
Patent No. 5,766,5201. 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.
At the present time, 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.
Cell cultures have become increasingly significant in economic and commercial
importance. The expression of
recombinant products in cell culture systems is becoming widespread and has
greatly increased the number of
products available for use in industry and medicine. Practical incorporation
of these cultures into commercial products
has been hampered by their fragile nature and the special requirements in
handling them.
The conventional approach for protection of biological materials during
desiccation is based on the water
replacement hypothesis, first introduced by Webb ("Bound Water in Biological
Integrity". Springfield, IIL, C. C.
Thomas, 19651. Crowe, Roser and their collaborators, as well as several other
groups, reported that disaccharides
(sucrose and trehalose) are effective as protectors against desiccation-
induced damage because they effectively
replace "water of hydration" at the polar groups of biological molecules.
Bronshtein and Leopold (1996, Cryobiology
3316):626-7) showed that some minimal amount of disaccharides ("10 g per g
enzyme) is required to provide
macromolecule stabilization at high temperatures (37° and 50°
C1. These observations are in agreement with the
conventional belief that desiccation-induced damage results from an increase
in hydration forces between biological
molecules in the absence of protective fillers (i.e. sugars) and that cells
should be filled with significant amounts of
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sugar or other protectors to survive desiccation. Because most disaccharides
permeate slowly into cells, the
generation of sufficient concentrations of intracellular protectants remains a
significant challenge.
Accordingly, there is a need for a method of increasing the tolerance of
bacterial cells to desiccation in large,
industrial scale volumes. 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.
Summary of the Invention
A scaleable method for preserving bacterial cells in a dried state is
disclosed. The method 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.
In one variation, two or more of the fermentation parameters are modified to
enhance the cells' ability to
survive dehydration.
In another variation, 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. In a preferred variation,
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.
Brief Description of the Drawings
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.
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Detailed Descriution of the Preferred Embodiment
Others have shown that 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. Similarly,
the use of nutrient starvation, heat shock and "osmoadaptation" by salt
addition resulted in higher survival of
Pseudomonas flourescens preserved by desiccation at 20° C. Legg
suggested that shock related production of
intracellular trehalose is responsible for the increase in cell survival in
his experiments. Tunnacliff et al. (WO
98124882) also indicated that expression of trehalose production genes is a
solution for bacteria survival in the dry
state. Other modifications in fermentation protocols focus primarily on
increasing the osmotic strength of the medium
to increase the accumulation of internal osmotic protectants such as
trehalose, glutamate and betaine. This
accumulation results from the addition to salts from 0.0-0.6 M salts to
present an osmotic stress to the cells.
Glaasker et al. 1996 Osmotic regulation of intracellular solute pools in
lactobacillus plantarum. J. Bacteriol. 178:575
582; Horlacher et al. 1996 Characterization of a cytoplasmic trehalose of
Escherichia coli. J. Bacteriol. 178:6250
6257; Ogahara et al. 1995 Accumulation of glutamate by osmotically stressed
Escherichia coli is dependent on pH. J.
Bacteriol.177:5987-5990.
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. cremoris ATCC 19257;
Lactic acid bacteria, Lactobacillus
acidophilus ATCC 4356, catfish vaccine, attenuated Edwardsiella ictaluri;
kennel cough in dogs, attenuated Bordetella
bronchiseptica; strangles in horses, attenuated Streptococcus equi;
salmonellosis, attenuated Salmonella choleraesuis;
and others. The common feature of the organisms preserved utilizing
fermentation modification and subsequent
preservation by foam-drying, is accumulation of "internal protectants",
including carbohydrates and peptides that
support viability upon drying. The modification of fermentation serves to
increase the production and accumulation of
these compounds through the means herein described. The assumption of this
process is that the organisms have the
genetic capability to produce such "internal protectants". The modifications
of fermentation described herein are only
applicable if the genetic potential for expression of internal protectants is
demonstrated. If the organisms do not have
the genetic capability for such protectants, the genes responsible can be
provided for these organisms. The genetic
transformation, or induction of transferred genes, may be accomplished by
standard techniques. Preferably, the
transferred gene(sl are expressed only in response to the modified
fermentation techniques disclosed herein.
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
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(phyto- and zoo-1, 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
andlor bioassays that require indicator
cells, as well as any pre-packaged recombinant expression systems that use a
defined cell line.
Foam-Dryin4 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.
A variety of 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.
More particularly, 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.
In one preferred variation of the present invention, 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. Among the non-reducing ketoses, are
included: the six carbon sugars, fructose,
sorbose, and piscose; the five carbon sugars, ribulose and xylulose; the four-
carbon sugar, erythulose; and the three-carbon
sugar, 1,3 dihydroxydimethylketone. Among the methylated monosaccharides, are
the alpha and beta methylated forms of
gluco, manno, and galacto pyranoside. Among the 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. In addition,
sucrose andlor other fillers may be effectively transformed into a stable foam
composed of thin amorphous films of the
concentrated sugar when dried under vacuum.
Combining monosaccharides with disaccharides and oligosaccharides effectively
prevents crystallization of the
oligosaccharides during dehydration. 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. For example, polysaccharides, like
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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.
To facilitate scale-up of the preservation process to accommodate commercially
useful volumes, 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.
In a variation of the present invention, 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. In other words, 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.
While low vacuum pressures (in the range of 0.1-0.9 atm) may be applied to
facilitate the initial evaporation to
produce a concentrated, viscous cell suspension, 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. Such
"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~o 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 farm.
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 Nz atmosphere
andlor chemical desiccant, prior to subsequent
processing operations, (e.g. stability drying, vitrification or millingl.
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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:
(1) A mixture (100 II containing 50 I of 50% by weight sucrose and 50 I of an
ice nucleating bacteria
suspension, pNB) Pseudomonas syringae ATCC 53543, were placed in 1.5 ml
plastic tubes and preserved by drying at
room temperature. First, the samples were dried for 4 hours under low vacuum
(0.2 atml. Second, the samples were
boiled during 4 hours under high vacuum ( < 0.01 atml. After boiling under
high vacuum, a mechanically-stable porous
structure was formed. Third, the samples were stored during 8 days over
DRIERITE under vacuum at room temperature.
After 8 days, the samples were rehydrated with 500 I water. Rehydration of the
samples containing the dry
foams was an easy process that was completed within several seconds. Then the
samples were assayed for ice nucleation
activity in comparison with control samples. There was no significant
difference between the ice nucleating activity per
1,000 bacteria in the samples preserved by the present method versus the
control samples.
(21 A sample containing a 1:1 mixture of a concentrated suspension of ice
nucleating bacteria (INB)
Pseudomonas syringae ATCC 53543 and sucrose has been used. The sample was
mixed until all sucrose crystals were
dissolved, so that the final suspension contained 50 wt% sucrose. The
suspension was placed in 20 ml vials at 2 g per
vial. The vials were dried inside a vacuum chamber. The vials were sitting on
the surface of a stainless steel shelf inside
the chamber. The shelf temperature was controlled by circulating ethylene
glycollwater antifreeze at a controlled
temperature inside the shelf. Before the vacuum was applied the shelf
temperature was decreased to 5 C. Then, the
hydrostatic pressure inside the chamber was decreased to 0.3 Torr. Under these
conditions the suspension boiled for 30
min. The temperature of the shelf was then slowly (during 30 mint increased up
to 25 C. Visually stable dry foams inside
the vials under these experimental conditions were formed within 3 hours.
Subsequently, the samples were kept under the
vacuum at room temperature for one more day. 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 can nucleate an ice crystal in a 10 I buffer drop
during 5 minutes at -5 C. The results of the assay
show ice nucleating activity in the preserved samples was equivalent to that
observed in fresh controls.
(3) A concentrated INB suspension was frozen to -76 C for future use. 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
glycollwater 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
25 C. Visually, the formation of stable dry foams inside the vials under these
conditions was completed within 2.5 hours.
After removal of several vials, the temperature was increased to 50 C and the
remaining samples were kept under vacuum
for 7 days.
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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 u1 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%1. 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.
(5) A 1.5 ml tube containing a frozen (-76 C) suspension of E. coli (XL10-
GOLD) from Stratagene was
thawed in an ice bath. A 100 I aliquot was transferred to 50 ml of NZYM
(Casein digest yeast extract medium) broth and
incubated at 37 C on an orbital shaker overnight. After 14 hours of growth, 10
ml of this growth culture was inoculated
into 100 ml of sterile NZYM broth to continue the culture growth at 37 C.
During the culture growth the optical density
(0D@620 nm) was measured every hour to determine the end of logarithmic
bacteria growth. When the transition phase
was reached (OD=1 to 1.06) the cells were ready to be harvested. 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 m1) 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 rehydration 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.
Moreover, the portion of surviving cells did not decrease during the
subsequent 24 days of storage under vacuum at room
temperature.
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
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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.
For any given culture to be preserved, the nature and stability
characteristics of the cells will determine the
maximum temperature they can withstand during the foam-drying and stability-
drying steps. In some cases for example, it
has been shown that after foam-drying at room temperature, 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. Similarly,
the temperatures and pressures to which foam-dried cultures are exposed may be
varied to provide a range of stability-
drying parameters. Again, cell viability serves as an indicator of optimal
stability-drying conditions. In some cases,
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. In a
preferred aspect, 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.
Based on empirical results obtained with dried 10 u1 drops of 15% sucrose +
15% raffinose solution, it was demonstrated
that more than 12 hours of stability drying at temperatures above 70 C was
required to raise Tg to above 25 C. Foam-
drying in these experiments was for 12 hours at room temperature (20 C). The
results suggest that extended stability-
drying times (more than 12 hours at 70 C and mare than 36 hours at 50 C) may
be needed to effect increases in Tg over
room temperature.
To ensure that the Tg is actually greater than the storage temperature, at
least two methods are known for
estimating Tg by thermal analysis. Differential scanning calorimetry (DSC) is
the most commonly used technique.
However, DSC may be unreliable for measuring Tg in samples that contain
polymers. Alternatively, Thermally Stimulated
Polarization Current (TSPC) methods are specifically adapted for analysis of
polymers. The TSPC method is preferred
because it is reliable for all samples, although it requires slightly larger
sample volumes.
After foam-drying and optional stability drying, the dried foams may be stored
at a selected storage
temperature for a selected storage period. Following drying andlor storage,
dried bacterial cultures can be rehydrated
with culture medium and assayed for viability. Typically, 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
_g_
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survival can be calculated by dividing the surviving CFUImI of the sample
being tested by the CFUImI of the original
fermentation.
Modification of Fermentation to Enhance Desiccation Tolerance - 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.l.
There are two major kinds of fermentation processes, traditional and
controlled. Traditional fermentation is
driven by the metabolic activities of the natural flora associated with the
raw materials. In controlled fermentation,
the desired microorganisms, so called "starter cultures", are added to the raw
materials and then the appropriate
environmental conditions are set. Industrial fermentation is exclusively
operated as controlled processes, where the
use of defined starter cultures increases the probability for success of the
process and ensures high consistency of
the product.
For the purposes of the present disclosure, microbial fermentation is
considered a multi-dimensional process,
comprising many parameters including; culture temperature, pH, osmotic
strength, time of cell growth (cell densityh
ionic strength, concentration of bivalent cations, and media composition
(e.g., nutrient, oxygen, nitrogen
concentration, etc.l. 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-dryingl, 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 shapel. 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
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of the growth curve increases in the low and high growth range, and becomes
vertical at inhibitory (Iethall
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
S 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.l, and "thermophiles" (growth range from 40° C to
above 100° C1.
The growth rate decreases rapidly at temperatures above or below the optimal.
In addition, there are
precise temperatures at which bacterial growth does not occur. When exposed to
increased temperatures, 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. In
contrast, minimum growth temperature is set by factors that cause weakening of
hydrophobic bonds involved in
higher levels of protein structural organization.
~H - Based on their response to external pH, bacteria are classified as
"acidophile" (grow best at low pHl,
"neutrophile" (grow best at neutral pH, like E. cold, and "alkalophile" (grow
best at pH above 7.0). 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.
Many different compounds can function as compatible 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. 45:569-6061. Some of the listed compounds are also widely
known as "fillers" (i.e., trehalose,
sucrose, manitol, glycerol, and glucitol).
Cell Density ("pheromones" or "autoinducers") - Many bacteria have developed
mechanisms for sensing and
responding to an increase in population density (Gary M. Danny & Stephen C.
Winans, Eds. 1999 Cell-Cell Signalinn in
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Bacteria, ASM Press, Washington, DC). High cell density, so called "quorum
sensing", is known to contribute to the
regulation of many important physiological processes. The development of
genetic competence, sporulation,
bioluminescence, conjugation, plant and animal pathogenesis, production of
bacteriocines and antibiotics, are some of
the processes regulated by cell-cell communication which occurs when the
bacterial population reaches high density.
Many different signaling molecules have been described, but it is generally
accepted that homoserine lactones (HSLs)
mediate cell-cell communication in Gram-negative bacteria and that different
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. Within the cell, 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 Uibrio, Streptococcus, Enterococcus,
Pseudomonas, Myxococcus, Bacillus,
Agrobacterium, Erwinia, Rhizobium, Xanthomonas, Staphylococcus, Lactococcus,
Lactobacillus, and Streptomyces.
Divalent Cations - The composition of the fermentation broth will include a
large number of divalent cations.
These cations are involved in a wide range of functions, ranging from
cofactors in enzymatic reactions to providing
covalent bonding sites in cell wall formation. For example, the addition of
0.1 % Ca" to L. acidophilus ATCC 4356
changes the colonial morphology from a mixture of "rough" and "smooth"
colonial forms to almost completely
"smooth" forms. "Smooth" colonies are composed of small bacilloid cells while
"rough" colonies are composed of
filamentous cells. It has been shown that the "smooth" form survives freeze-
drying at higher yields than the "rough"
form. Wright and Klaenhammer 1981 Calcium-Induced Alteration of Cellular
Morphology Affecting the Resistance of
Lactobacillus acidophilus to Freezing. Appl. Environ. Microbiol. 41:807-815.
The enhancement of cell desiccation tolerance by modifications of fermentation
conditions may be more fully
appreciated with reference to the following working examples:
Fermentations were performed using a New Brunswick Scientific Company BioFlo
2000 fermenter with a 2
L working capacity. The fermenter was equipped with a pH module to control pH
by the addition of acid or base to
the fermenting culture as necessary.
Preservation was carried out generally as described above by foam-drying. Mare
specifically, 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. After 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. After a mechanically-stable foam had formed, 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
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remained under vacuum at 45° C for 36 hours. The samples were sealed
under vacuum prior to being removed from
the vacuum chamber.
After storage for the indicated time periods under the indicated conditions,
cells were re-hydrated with
buffer and assayed for viability. (liable cell counts were determined by
serial dilution of the sample and plating on
appropriate agar. The plates were incubated and colony-forming units (CFU)
determined. Percent survival was
calculated by dividing the CFUImL of the sample being tested versus the CFUImL
of the original fermentation.
Example 1
Fermentation of Lactobacillus acidophilus (ATCC 4356) at Low pH
Lactobacillus acidophilus ATCC 4356 1L. 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.
Conventional fermentation calls for growth on complex media (Difco MRS broth +
0.05% cysteine) under
pH-regulation between 5.80-6.00 at 37° C. The fermentation is
anaerobic. The broth was inoculated with cells from
a frozen seed. The cells were fermented to stationary phase indicated by an
O.D. of 2.4. Preservation survival
following foam-drying and rehydration was less than 20%. Bacteria were
enumerated by plating on MRS+0.05%
cysteine agar and incubating for 48 h at 37° C under anaerobic
conditions.
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. (@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 4Ø 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.
Exam 1e 2
Effect of Growth Phase on Survival Yields of L. acidophilus
Cultures were started from a frozen seed. The cells were fermented in
MRS+0.05% cysteine broth at 37°
C. Bacteria were harvested in logarithmic phase of growth (defined by the mid
point in exponential increase in O.D.
(optical density by absorbance at 600 nm) and in stationary phase (defined by
no increase in O.D.1. The bacteria were
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preserved by foam-drying. Bacteria were enumerated by plating on MRS+0.05%
cysteine agar and incubating for 48
h at 37° C under anaerobic conditions.
The results illustrated in Figure 1, show that preservation yields for both
log and stationary phase cells was
40%. The first two time points (prior to the zero time point) show cell counts
prior to foam-drying and following the
drying process, respectively. Clearly, after drying, the cell counts remained
essentially stable throughout the 17 day
storage period at 37° C. The cell density was 4.2x108 CFUImL in the log
phase versus 1.0x109 CFUImL in the
stationary phase.
Examule 3
Effect of Ca'Z on Desiccation Tolerance of L. acidophilus
Bacteria were fermented in Difco MRS+0.05% cysteine broth with the addition of
0.1 % Ca'Z at a
temperature of 42° C. The pH was maintained at 5.80. Cells were
harvested in stationary phase as determined by a
stabilization of optical density. The bacteria were preserved by foam-drying.
Bacteria were enumerated by plating on
MRS+0.05% cysteine agar and incubating for 48 h at 37° C under
anaerobic conditions.
Preservation survival was 47-50%. Untreated cells had a survival if less than
40%. Bacteria fermented
under the previously described conditions at 37° C with added Ca'2
alone had preservation yields of about 50% and
were stable for 16 days at 50° C.
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 Iat 600 nm)
was greater than 2.60 and the pH of the culture was less than 4Ø 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 um
filter. Samples of the depleted supernatant were neutralized by the addition
of 1 M NaOH until the pH reached 7Ø
Cells were fermented in MRS+0.05% cysteine broth under pH regulation (pH=5.80)
at 37° C. Logarithmic
phase cells were treated with depleted supernatant by centrifuging the samples
and re-suspending the cells in a
depleted supernatant from a previous unregulated fermentation. The bacteria
were preserved by foam-drying.
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
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Examule 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'z 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%. As illustrated in Figure 2, the bacteria in the mixed
powder were stable for 86 days at 25° C.
Examule 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 mLl, deionized water to 1000 mL and
adjusted to pH 7.4. Fermentation pH
was adjusted to 7.1 by the addition of 1 N 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
centrifugation 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.
Preservation survival after drying was 10-30% when the cells were harvested in
late logarithmic phase or
early stationary phase (defined as the first 60 minutes of stationary phasel.
Preservation survival after drying was
highest three hours in to stationary phase. Preserved bacteria were stable for
200 days stored at 4° C.
Examule 7
Growth to Stationary Phase of Bordetella bronchiseptica
B. bronchiseptica cultures were fermented by on Dextrose Starch broth at
37° C under aerobic conditions.
Dextrose Starch Broth is composed of dextrose (2.0 g1, soluble starch (10.0
g1, NaCI (5.0 g1, disodium phosphate (3.0
g), gelatin (bacteriological) (20.0 g1, glycerol (10.0 g), sodium acetate
(0.08 g1, 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. Survival after drying was
greater than 90%. (liability of the dried
samples was stable for over 70 days at 37° C. Parallel cultures were
dried by conventional freeze-drying. The results
illustrated in Figure 3 show that cells preserved by freeze-drying rapidly
loose their viability upon storage at 37° C in
comparison to cell preserved by foam-drying.
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Example 8
Fermentation to Stationary Phase of Lactococcus lactis cremoris DSM Strain
Bacteria were fermented in a skim milk-based medium at 30° C. The
fermentation pH was maintained at
5.80 by the addition of 4.76% NH40H. The fermentation was harvested at
stationary phase as defined by 90 minutes
S after the last 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. The
cells were preserved with a yield of 100%.
As illustrated in Figure 4, the preserved bacteria were stable for 108 days at
25° C and 29 days at 37° C.
Example 9
Effect of Osmotic Pressure on Desiccation Tolerance of
Lactococcus lactis subsp. cremoris ATCC 19257
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% NH40H. 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%.
25
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