Note: Descriptions are shown in the official language in which they were submitted.
W O 94/25564 PCTIGB94/00811 ~6~220
VIABLE BACTERIA
This invention relates to a process for the preparation of
compositions comprising dried microbial cells in a stasis state, to such
compositions and to living cultures prepared therefrom.
Storage of viable cultures is a recognised problem in the art. For
example, US 3,897,307 discloses (i) the use of a comblnation of an
ascorbate compound and a glutamate or aspartate as stabiliser for lactic
acid producing bacterial cells and (ii) the use of certain sugars,
particularly inositol at a concentration of 25mg/ml sample solution, as a
cryoprotectant where such bacterial cells are freeze-dried.
Mugnier et al, Applied and Environmental Microbiology, 1985,
pp 108-114 discloses the use of polysaccharide gels in combination with
certain nutritives, eg Cl 3 and C6 12 compounds, as the matrix for
freeze-dried bacterial cells. We have found that gel-forming
polysaccharides do not collapse on freeze-drying.
Redway et al, Cryobiology, 1974, Vol 11, 73-7-9 examined certain
monosaccharides (concentrations up to 150mg/2 ml sample) and related
compounds as media for long-term survival of freeze-dried bacteria.
We have now found that where microbial cells are suspended in a
certain matrix as hereinafter defined and dried under certain conditions
the short-term viability thereof is improved and that where such dried
systems are stored and rehydrated under certain conditions as hereinafter
defined the long term viability of the microbial cells is improved.
We have further found surprisingly that collapse of the matrix in
which the microbial cells are suspended does not lead to poor short-term
viability.
According to the first aspect of the present invention there is
provided a stabilised dried composition comprising microbial cells in a
stasis state suspended in a collapsed matrix.
By "stabilised" we mean that the degradation of the microbial cells is
reduced (which degradation would lead to a loss of recoverable viable
cells).
By "stasis state~ we mean that the cells are not metabolising,
dividing or growing (but are recoverable if subjected to a suitable
treatment).
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216 1~ 2 -
sy l~recoverable~l we mean cells which on exposure to suitable
conditions (ie rehydration and source of nutrient) are capable of growth
and division.
sy ~viable cells" we mean cells which on exposure to suitable
conditions (ie rehydration and source of nutrient) are capable of growth
and division.
By ~collapsed'~ we mean
i) that the matrix has shrunk and become less porous allowing little
penetration of low MW diffusive species into the matrix, eg it absorbs
little water vapour on exposure to humid air; and/or
ii) the matrix has experienced a temperature above its glass transition
temperature (Tg) such that viscous flow thereof has occurred leading to a
substantial reduction in surface area/volume ratio and encapsulating the
cells in a low porosity protective coating.
According to the second aspect of the present invention there is
provided a process for the preparation of a stabilised dried composition
comprising microbial cells in a stasis state suspended in a matrix which
process comprises the steps of:
A: mixing the microbial cells with an aqueous composition comprising the
material from which the matrix will be derived;
B: drying the mixture under conditions such that viscous flow of the
material occurs and the matrix collapses but does not unduly damage the
cells.
Preferably the composition prepared in Step B is stored at a
temperature below the Tg of the matrix, ie the composition has a Tg above
its anticipated storage temperature.
Accordingly, the composition prepared in Step B is preferably dried
further, so-called ~secondary drying", to increase the Tg of the matrix
such that the composition is stabilised to a broader range of storage
conditions, ie it can be stored at a higher temperature.
The microbial cells of which the stabilised dried composition
according to the present invention is comprised are preferably bacterial
cells. However, we do not exclude the possibility that alternative
microbial cells may be used, eg fungi, yeast, etc. Where the microbial
cells are bacterial cells they are preferably Gram-negative bacterial cells
although we do not exclude the possibility that they may be cells of a
W O 94l25564 PCT/GB94/00811
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Gram-positive bacteria. As examples of such Gram-negative cells may be
mentioned inter alia Pseudomonas fluorescens, Escherichia coli and
rhizosphere-associated bacteria.
The concentration of the microbial cells in the mixture prepared in
Step A of the process according to the present invention is between lO /ml
and lO /ml and preferably is between lO /ml and lO /ml.
The material which is mixed with the microbial cells in Step A of the
process according to the present invention is a polyhydroxy compound, eg a
polyol such as mannitol, inositol, sorbitol, galactitol, or preferably a
carbohydrate, more preferably a saccharide.
Where the material is a saccharide it may be a di-saccharide, a
tri-saccharide, an oligo-saccharide, or preferably a monosaccharide. As
examples of mono-saccharides may be mentioned into alia hexoses, eg
rhamnose, xylose, fructose, glucose, mannose and galactose. As examples of
disaccharides may be mentioned inter alia maltose, lactose, trehalose and
sucrose. As an example of a trisaccharide may be mentioned raffinose. As
examples of oligosaccharides may be mentioned maltodextrins.
The concentration of the polyhydroxy compound used in the mixture in
Step A of the process according to the present invention is between
lOmg/lO and lOOOmg/lO cells and preferably between 200mg/lO cells and
400mg/lO cells. The skilled person will be able to find by simple
experiment the concentrations from which a collapsed matrix can be prepared
for a particular polyhydroxy compound. For example, we have found that
inositol has an optimum concentration at about 45 mg/ml, it causes massive
cell damage above 60 mg/ml and does not collapse at below about 25 mg/ml.
Certain of the polyhydroxy compounds exhibit protective properties
over a wide range of concentrations, whereas certain others above a
critical concentration, which appears to be related to the solubility of
the polyhydroxy compound in the aqueous medium, exhibit a detrimental
effect.
The present invention is further illustrated by reference to the
accompanying drawings which illustrate, by way of example only,
compositions according to the present invention.
In the drawings:
Figure l illustrates in the form of a graph the variation of viability
of Pseudom~nAs fluorescens with inositol (additive) concentration when
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freeze-dried from water or 0.04M MgSO4. The vertical axis represents
viability in parts per billion (ie lE+09=lO ppb, lE+08=lO ppb, etc) and the
horizontal axis represents the concentration of the additive in milligrams
per sample. The black squares (~) on the graph plot inositol in magnesium
sulphate and the circles (o) plot inositol in water.
Figures 2 to 7 illustrate in the form of graphs the variation of
viability of freeze-dried PselldomonAs fluorescens with monosaccharide
concentration for a range of monosaccharides. The vertical axis represents
viability in parts per billion, in numbers of billions (ie lE+0=l billion,
5E-l=0.5 billion, 2E-l=0.2 billion, etc) and the horizontal axis represents
the sugar concentration in milligrams per sample. The monosaccharides
illustrated are:
Figure 2 galactose Figure 5 xylose
Figure 3 fructose Figure 6 rhamnose
Figure 4 glucose Figure 7 mannose
Figure 8 illustrates in the form of a graph the variation of cell
death rate (kl) with Tg of the matrix and the variation of Tg with relative
humidity. The left-hand vertical axis represents kl, the right-hand
vertical axis represents Tg (C) and the horizontal axis represents
relative humidity (~). The black squares (~) on the graph connected by a
solid line plot the cell death rate constant (kl) and the lozenges
connected by a broken line plot the glass transition temperature (Tg).
In the drawings, viability is expressed as the number of viable
bacteria per lO viable bacteria in the original suspension, ie it
represents the number of bacteria which survive from each one billion
bacteria which were viable initially. It is represented as the parts per
billion viability (ppb), ie lO ppb is equivalent to lO0~ survival and lO
is equivalent to lO~ survival, etc.
From Figure l it can be seen that at low inositol concentrations the
viability of the cells is maintained whereas at higher concentrations, eg
greater than lOOmg/sample (equivalent to 50mg/ml), cell viability rapidly
decreases.
W O 94n5564 21 6 12 2 0 PCT/GB94/00811
From Figures 2 to 7 it can be seen that certain monosaccharides
protect the cells from damage at low concentrations, eg less than lO
mg/sample (equivalent to 5mg/ml) and that protection is substantially
maintained at high concentrations, eg about 400 mg/sample (equivalent to
130mg/ml).
From Figure 8 it can be seen that where the Tg drops below the storage
temperature (represented by the top horizontal line at 21-22C), the rate
of cell death (kl) increases significantly, illustrating the importance of
maintaining a glassy state during storage.
The concentration at which the material is effective, ie it collapses
without unduly damaging the cells, is dependent on a variety of factors,
including inter alia: the volume fraction of the cells in the suspension in
Step A; the inherent glass transition temperature of the polyhydroxy
compound; the variation in glass transition temperature of the matrix as a
function of water concentration therein; and the temperature to which the
matrix is exposed during and after freeze-drying.
It will be appreciated that the polyhydroxy compound may act as (i) a
cryo-protectant at low temperature, particularly against damage by
ice-particles during freeze-drying; and /or (ii) a lyo-protectant
protecting against damage due to loss of water during drying and/or
storing; and /or (iii) a nutrient source during recovery of the cell.
The microbial cells for use in the process of the present invention
may be grown in conventional growth media, eg nutrient broth or tryptone
soya broth. They may be harvested at any convenient phase of growth,
preferably at early stationary phase.
For example, a culture is grown in or on a suitable medium, eg liquid
or solid plates, to give a desired cell concentration. The cells are
isolated, typically by centrifugation. They are resuspended in an aqueous
composition comprising the material which will form the matrix and
optionally certain other additives as mentioned hereinafter.
Preferably, the microbial cells used in the process of the present
invention are isolated from the growth medium, resuspended in a solution
comprising polyhydroxy compound, suitable additives, etc and dried.
However, we do not exclude the possibility that the polyhydroxy compound
and suitable additives, etc are added to the cells in the growth medium and
the resulting mixture dried.
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Where the microbial cells are resuspended, they are resuspended in a
suitable aqueous medium, eg aqueous MgS04 solution, or preferably water,
containing the polyhydroxy compound.
The drying in Step B of the process according to the present invention
may be carried out by, for example, evaporation, vacuum-drying,
spray-drying, air-drying or preferably freeze-drying.
As hereinbefore defined it is essential to achieve viscous flow during
at least the drying step, Step B, or any subsequent step.
Typically the water content of the dried composition prepared in Step
B is less than 15~ w/w.
Where the drying in Step B comprises freeze-drying the composition
typically contains one or more suitable additives. As examples of suitable
additives may be mentioned inter alia cryo-protectants, for example sugars
or polymeric species, eg polyvinylalcohol, polyvinylpyrrolidone;
lyo-protectants, for example sugars or polymeric species, eg polyvinyl
alcohol, polyethylene glycol; or preferably anti-oxidants or so-called
potentiators, eg ascorbate or glutamate. We do not exclude the possibility
that other additives may be present, for example, so-called bulking agents,
for example crystallising sugars, eg mannitol, and osmo-regulants, eg
betaine, urea/trimethylamine-N-oxide~ proline, sarcosine.
The present invention is further illustrated by reference to the
following Examples.
EXAMPLES 1-6
These Examples illustrate compositions according to the present
invention wherein the matrix comprises rhamnose.
Pse~d~on~ fluorescens was cultured in standard media (double
strength nutrient broth~ and harvested in early stationary phase by
centrifugation. The cell concentrate was resuspended in sterile water and
a sufficient volume of an autoclave-sterilised, concentrated rhamnose
solution was added to give approximately 200 aliquots of a final
concentration of 200 mg of sugar to 2x101 cells in a total volume of 4ml
water in 5ml capacity freeze-drying vials.
The vials were loaded onto the temperature-controlled shelves of a
freeze-drying apparatus and the shelf-temperature was driven to -30C,
freezing the contents of the vials and lowering their temperatures to -28OC
to -30OC, over a two hour period. Vacuum was applied and primary drying
~ W O 94125564 21612 2 0 PCT/GB94100811
was carried out over a period of 48 hours. The shelf temperature was
raised to 0C and secondary drying was allowed to occur for 24 hours. The
vials were brought to room temperature and sealed under vacuum before
removal from the freeze-drier.
The vials were stored at 4C in vacuo (Example l) or in humidity
controlled air at 21C (Examples 2-6).
The samples were rehydrated in sterile water and viable bacterial cell
numbers were determined by serial dilution in water followed by plating
onto King's B growth medium. The number of colony-forming units (cfu) on
the highest dilution plates was used to calculate the number of bacterial
cells per unit volume which survived the freeze-drying and storage
conditions.
Immediately after freeze-drying the viability of the cells was
3 1o8 b
The results from bacteria stored at 4C in vacuo are shown in Table l.
TABLE l
Example Viability (ppb)
No.
after storage for (weeks)
3 50
l ND 5xlO
CTl o
ND: not determined
CTl is a Comparative Test with no rhamnose present.
From Table l it can be seen that the presence of a rhamnose matrix
improves the viability of the bacteria substantially.
The results from bacteria stored at 21C in controlled humidity
chambers are shown in Table 2.
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T~3LE 2
Example Rel Viability (ppb) r
No. humidity after storage for (days)
(RH~) 13 27 34
2 1 8xlo 3xlO 4xlO
3 4 3xlO 7xlO 2xlO
4 9 9xlO 5xlO 4xlO
23 4xlO 4xlO 8xlO
6 44 3x107 lxlO 3xlO
CT2 1 10
CT2 is a Comparative Example with no rhamnose present.
From Table 2 it can be seen that the presence of a sugar substantially
increases the viability even at low RH, ie Ex 2 compared with CT2.
EXAMPLES 7-10
These Examples illustrate compositions according to the present
invention wherein the matrix comprises rhamnose and magnesium sulphate.
The procedure of Examples 1-6 was repeated except that the cell
concentrate was resuspended in 0.04M magnesium sulphate instead of sterile
water and rhamnose solution was then added.
The viability of the cells imm~;Ately after freeze-drying was 5xlO .
The freeze-dried cells were stored at the temperatures and for the
periods of time shown in Table 3.
TABLE 3
Example Storage Viability (ppb) after days
No.temp (C~ 50 100 175 365
7 -20 5x1o8 5x1o8 5xlO 5xlO
8 4 2xlO 2xlO lxlo
9 15 " lxlO 5xlO
" 2xlO
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It can be seen from Table 3 that the formulation provides substantial
protection over a range of temperatures.
s EXAMPLES 11-15
These Examples illustrate compositions according to the present
invention wherein sodium ascorbate and sodium glutamate are present in the
matrix.
The procedure of Examples 1-6 was repeated except that concentrated
aqueous solutions of sodium ascorbate and sodium glutamate were added to
the resuspended cells in water and rhamnose.
The viability of the cells imme~i~tely after freeze-drying was 2xlO
ppb.
The samples were stored in humid air at 21C.
TA~3LE 4
Example Relative Viability (ppb) Tg
No. humidity after storage for (days) C
(RH~) 34 128
11 0 1xlo8 lx1o8 25
12 14 '~ ~ 24
13 30 " 4xlO 14
14 40 9xlO 5xlO 12
53 3xlO 3xlO 8
From Examples 11 and 12 in Table 4 it can be seen that storing the
dried cells at temperatures below the Tg of the matrix stabilises the cells
for long periods.
The results indicate that the combination of collapsed matrix in a
glassy state and the presence of an anti-oxidant provides a matrix which
can stabilise the viable cells for long periods of time under relatively
harsh environments.
EXAMPLE 16
A 4g pellet of Pseudomonas fluorescens cells, separated from culture
media by centrifugation and ccnt~ining 5.10 viable cells, was mixed with
14g of a commercial grade of maltodextrin (Glucidex IT19) and 1.6g of
sodium ascorbate and the material, initially at room temperature, dried
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2~L~122 - 10 -
under reduced pressure, in which the evaporated water rond~n~ed onto an ice
trap maintained at -50C.
Drying was terminated after approximately 18 hours, at which time the r
vacuum was of the order of 1 mbar.
Samples rehydrated into pure water and plated onto Nutrient agar
typically showed 50-90~ recovery of viable cells.
Materials prepared by this method appear as collapsed amorphous
matrices with glass transitions exceeding 20C (when samples were exposed
to standard laboratory relative humidities).
