Note: Descriptions are shown in the official language in which they were submitted.
CA 022~4431 1998-11-24
Prevention of Irreversible Aggregation of Viable Microorg~ni~m~ upon Drying
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method and composition for preventing
5 irreversible aggregation of viable microorg~ni~m~ and other biological agents which
exhibit aggregative activity upon drying.
2. Prior Art
Commercial products cont~ining viable microorg~ni~m~ as the active
ingredients are often sold on the basis of colony forming units (cfus). Each of the
10 viable microbes such as a fungal spore or a bacterium is capable of producing one
cfu. However, if more than one microbes form aggregates in the finished productsand cannot be disassociated during application, one aggregate will produce one cfu
only, even though all the microbes in the aggregate are viable. Such aggregation is
often referred to as irreversible aggregation, and it is undesirable in formulations of
15 commercial products, because it can dramatically reduce product titer and thus
increase manufacturing cost.
Irreversible aggregation of microorg~ni~m~ is a problem often associated
with formulation processes of microbial products, particularly with the processes
involving drying such as air drying, spray drying and freeze drying. Irreversible
20 aggregation caused by drying can easily be identified by visual and microscopic
ex~min~tions as showing increase in particle size, quicker settling in suspension,
and more than one viable microbes in a single aggregate.
Depending on the microorganisms and the fermentation methods used, not
all the microbes exhibit irreversible aggregation to the same magnitude upon
25 drying. It has been found that microbes produced by submerged fermentation and
thereafter dried in a concentrated form are often subject to irreversible aggregation.
The problem of irreversible aggregation becomes more pronounced with fungi that
produce air-dispersal spores in nature. A great number of fungi produce
air-dispersal spores, including many commercially important species of Aspergillus,
30 Beauveria, Metarhizium, Neurospora, Penicillium, and Trichoderma. The surface
~ , ., ~ .
CA 022~4431 1998-11-24
of air-dispersal spores is covered by a hydrophobic layer, known as "rodlets". The
rodlets, however, are absent on the surface of the spores which are produced in
submerged fermentation. It was also found that the spores without rodlets form
irreversible aggregation upon drying, while spores with rodlets do not form
5 irreversible aggregates under the same drying conditions after being pre-wetted in
water in the presence of surfactants. It seems that the rodlet layer plays a role in
preventing spores from aggregation upon drying. In the absence of a rodlet layer,
the spores have direct surface contact, the amorphous cell wall materials (possibly
polysaccharides and proteins) on the spore surface may become denatured and
10 insoluble due to drying and the spores are bonded together as irreversible
aggregates.
The use of viable microorg~ni.cm~ as agricultural products often face the
problem of lack of efficient delivery systems. The challenge is to formulate viable
microorg~ni~m~ into a stable and useful form that meets the current agricultural15 practice. Although fresh culture and frozen microbe concentrate are easy-to-use
formulations that have been used for some microbial products, the short shelf-life
and restricted storage conditions of such formulations limit the product distribution.
It would be ideal that viable microorg~ni~m.c can be stored and delivered in dryforms and be readily dispersible in cold water. This would mean that packages of20 dried formulation could be reconstituted quickly (within 5 minutes) in cold water
by the end users in the field as needed. The development of such dry formulations
has been hampered by irreversible aggregation of active ingredients upon drying.In addition to loss of titer, the irreversible aggregation also causes low dispersibility
and faster settling in water which are undesirable physical properties of water
25 dispersible formulations for agricultural use.
A method for encapsulating biological material is described in Baker et al.,
U.S. Patent 5,089,407. According to this patent, beads cont~ining the biologicalmaterial are formed using an aqueous nonionic polymer solution. However, the
dried beads that are formed dissolve slowly in cold water and may require more
30 than 30 minutes to completely dissolve. This makes such formulations undesirable
as ready-to-use delivery systems in the field.
CA 022~4431 1998-11-24
Another encapsulation process for microorg~nismx is described in Chen et
al., U.S. Patent 5,290,693. This involves the use of polyvinyl alcohol to form
beads which immobilize the microorg~ni.cm~. However, once again the beads
cannot be quickly dissolved in cold water.
It is the object of the present invention to develop a solution to the
particular problem of irreversible aggregation of fungal spores upon drying as well
as the broader problem of microorg~ni.~m~ in general which tend to irreversibly
aggregate upon drying.
SUMMARY OF THE INVENTION
The present invention in its broadest aspect relates to a method for
preventing irreversible aggregation of viable microorg~ni.cm~ which exhibit
aggregative activity upon drying. The method comprises dispersing the
microorg~ni~m.~ in an aqueous polymer solution cont~ining a water-soluble or
water-dispersable low molecular weight polymer such that the polymer solution
15 inhibits direct surface contact between the individual microorg~ni~m~. The polymer
solution cont~ining the dispersed microorg;lni~m~ is then dried to form a dry
continuous polymer matrix cont~ining the dispersed microorg~ni~m~. This dry
continuous polymer matrix is adapted to substantially totally dissolved within five
minutes in cold or room temperature water and to thereby provide a reconstituted20 solution cont~ining uniformly dispersed microorg~ni~m.c.
The main advantages of the present invention are: (1) it can effectively
prevent irreversible aggregation of microorg~ni.~mc upon drying and improve
product titer recovery by 2 to 50 fold; (2) it enables formulation with high titers
loading (higher than 1 x 10'~ cfus/g dry product) and therefore the products can be
25 delivered in a concentrated form; (3) the finished dry products have very high rates
of solubility or dispersibility in water, which allows viable microorg~ni~m.c to be
formulated as ready-to-use water-dispersible powders, pellets or granules; and (4)
the process is simple and readily adaptable to commercial scale of freeze drying,
spray drying and air drying processes.
According to a preferred feature, the invention relates to a method for
preventing irreversible aggregation of viable microorg~ni.~m~ which exhibit
aggregative activity upon drying, which comprises mixing said microorg~ni~m.c in
CA 022~4431 1998-11-24
an aqueous polymer solution containing a water-soluble or water-dispersible low
molecular weight polymer so as to uniformly disperse the microorg~ni~m~ within
the polymer solution such that the polymer solution inhibits direct surface contact
between individual microorgani~m~, said polymer being either a water-soluble
5 polymer selected from the group consisting of polyvinyl pyrrolidone having a
molecular weight under 40,000, polyethylene glycol having a molecular weight
under 8,000 and water-soluble proteins or a water-dispersible polymer selected
from the group consisting of skim milk and plant-derived proteins, spreading themixture of polymer solution and dispersed microorgani~m~ thus obtained as a layer
10 on a flat surface, and drying the layer while resting on the flat surface to form a
dried continuous polymer matrix cont~ining dispersed microorg~ni.~m~, said driedcontinuous polymer matrix being adapted to substantially totally dissolve within 5
minutes in cold or room temperature water and provide a reconstituted solution
cont~ining uniformly dispersed microorgani~m~. Preferably, the dried layer that is
15 obtained is crushed or ground into granular or powder and stored in sealed
packages.
The above method is applicable to many different microorg~ni~m~,
including fungi, bacteria, viruses, nematodes and plant and animal cells. The fungi
are typically yeast and propagules of filamentous fungi, e.g. asexual and sexual20 spores and mycelium. The asexual spores may be conidiospores of
Deuteromycetes, such as species of Aspergillus, Beauveria, Metarhizium,
Neurospora, Penicillium and Trichoderma. These may include Beauveria bassiana,
Penicillium bilaii and Trichoderma harzianum.
The invention also relates to a readily water-dispersible microorganism
25 composition in the form of dry powder or granules comprising viable
microorgani~m.~ which exhibit aggregative activity upon drying, said
microorg~ni.~m.~ being dispersed in a dried continuous polymer matrix formed of a
low molecular weight water-soluble or water-dispersible polymer such that the
matrix prevents direct surface contact between the individual microorg~ni.~m~, said
30 polymer being either a water-soluble polymer selected from the group consisting of
polyvinyl pyrrolidone having a molecular weight under 40,000, polyethylene glycol
having a molecular weight under 8,000 and water-soluble proteins or a water-
CA 022~4431 1998-11-24
dispersible polymer selected from the group con~ ting of skim milk and plant-
derived proteins, the dry powder or granules having been formed by drying a
mixture of said microorg~ni~m.~ uniformly dispersed in an aqueous solution of said
polymer in the form of a flat layer and forming powder or granules from the dry
flat layer, in which the solid polymer matrix is substantially totally soluble within 5
minutes in cold or room temperature water.
The products of the present invention are particularly valuable for use with
viable microorg~ni.~m~ used in agriculture. Thus, they are capable of being stored
at ambient temperatures for periods of time of up to one year without losing their
10 activity and are also capable of being easily and quickly dissolved or dispersed in
cold or room temperature water. They can be dissolved or dispersed within five
minutes, and in some cases within one minute, in ambient (cold or room
temperature) water.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA is a photomicrograph at 90,000 x m~gnification showing the
presence of rodlets on the surface of a conidiospore of Penicillium bilaii;
Figure lB is a photomicrograph at 90,000 x m~gnification showing the
absence of rodlets on the surface of a conidiospore of Penicillium bilaii produced
by submerged fermentations;
Figure 2 represents irreversible aggregation of microbes upon drying and the
concept for preventing the irreversible aggregation in the present invention;
Figure 3A is a photomicrograph showing freeze dried polymer matrix
having entrapped conidiospores of Penicillium bilaii. The size of the spores ranges
from 1.5 to 4 microns;
Figure 3B is a cross-sectional photomicrograph showing that the
conidiospores of Penicillium bilaii are entrapped separately in the polymer matrix
depicted in Figure 3A;
Figure 4 is a photomicrograph showing spores released after the polymer
matrix depicted in Figure 3A is dissolved in water;
Figure 5 represents particle size distributions of a liquid spore concentrate ofPenicillium bilaii before and after freeze drying in skim milk - sucrose matrix;
CA 022~4431 1998-11-24
Figure 6 represents particle size distributions of a liquid spore concentrate ofTrichoderma harzianum before and after freeze drying in PVP or skim milk -
sucrose matrix; and
Figure 7 represents particle size distributions of a liquid spore concentrate of5 Beauveria bassinana before and after freeze drying in PVP or skim milk - sucrose
matrix.
DETAILED DESCRIPTION OF THE INVENTION
An example of air-dispersed spores covered by rodlets is shown in Figure
lA, this being a conidiospore of Penicillium bilaii. Figure lB shows that the
10 rodlets are absent on the surface of a conidiospore of Penicillium bilaii produced
by submerged fermentation.
The principle of this invention is shown in Figure 2. Viable
microorg~ni~m~ are first admixed with an aqueous solution cont~ining sufficient
amounts of water-soluble or water-dispersible polymers to block direct surface
15 contact between individual microbes. The mixture is then subject to a drying
process to remove unbounded water, which can be either freeze drying or spray
drying or simply air-drying on a flat surface. Upon drying, the polymer solutionbecomes a continuous matrix where the microbes are entrapped and unbounded
from each other. The dry polymer matrix can be any physical forms such as
20 powder, pellets or granules depending on the drying process and post-drying
process used. When reconstituted in water or in aqueous solutions, the polymer
matrix is dissolved and the microbes entrapped are dispersed in the reconstitutesolution for application. Irreversible aggregation upon drying initiated by direct
surface contact between individual microbes can, therefore, be effectively
25 prevented.
Water-soluble or water-dispersible polymers useful in this invention can be
synthetic or natural polymers and can be ionic or nonionic polymers. Since the
polymer composition must be capable of blocking surface contact between
microbes at high loading rates, sufficient polymer must be present to form a
30 continuous matrix where microbes are trapped and separated from each other.
Polymers used should be capable of providing solutions of at least 10% by weightin water at temperatures below 30~C. Typical exarnples for water-soluble polymers
~ .... . .
CA 022~4431 1998-11-24
useful for this invention are polyvinyl pyrrolidone (PVP) with molecular weight
under 40,000, polyethylene glycol (PEG) with molecular weight under 8,000 and
water-soluble proteins such as bovine serum albumin (BSA). Typical examples
for water-dispersible polymers are skim milk and proteins derived from plants such
5 as soybean. Skim milk has been found to be particularly useful because of its low
cost, ready availability and ease of use. It is most conveniently used in the form of
instant skim milk powder.
The dry polymer matrix produced by the present invention must provide
quick release of the microbes upon reconstitution in water. Thus, the polymer used
10 should have high rates of solubility or dispersibility in cold or room temperature
water for broad agricultural applications such as foliage spray and seed inoculation.
To increase solubility of the final dry products, the polymers can be used alone or
in combination, or used with other compounds such as sugars, salts, and amino
acids. Sugar compounds such as glucose, fructose, sucrose, lactose and trehalose15 can improve the solubility of spray dried or freeze dried matrixes of skim milk or
other proteins.
A number of conventional additives may also be incorporated into the
polymer matrixes of the present invention. Such additives include, but are not
limited to, preservatives, stabilizers, protectants, colouring agents and surfactants.
20 Crosslinked polymers or cro~linking agents which cause the formation of covalent
bonds of the polymer in the matrixes, however, should not be used. Polyvalent
cations which cause strong ionotropic gelation should also be avoided.
Polymers and other compounds used in the present invention must be
compatible with the microbes to be encapsulated. The compatibility between the
25 polymers and the microbes can be easily established by those skilled in the art.
Most microbes are subject to cytoplasm and structural damage from drying
and freezing processes, particularly when the processes involve extreme
temperatures such as the high temperatures in spray drying and low temperatures in
freeze drying. The mech~ni~m~ that cause drying and freezing damage in
30 microorg~ni~m~ are not yet fully understood. Although it has been suggested that
in fungi the spores are less sensitive to drying and freezing damage than mycelium,
and in bacteria the gram positive species are less sensitive than gram negative
CA 022~4431 1998-11-24
species, it is more likely that each microorg;~ni~m~ needs to be treated as a special
case along with the drying and freezing processes used. Addition of protectants
such as sugars, polyhydroxy alcohols, dimethylsulfoxide and amino acids described
in International Patent Publ. No. WO 93/00807 can reduce cell damages from
drying and freeze drying. Appropriate protectants for a specif1c microorganism can
be selected empirically by those skilled in the art.
Other factors such as method of fermentation, age of culture, rate of growth,
and type of growth medium as described in U.S. Pat. No. 5,288,6340 can also be
manipulated to produce microbes that are less sensitive to drying and freezing
1 0 damages.
To prepare the polymer matrix, a number of process schemes can be
followed. The polymer and other additives can be either admixed in fermentation
broth cont~ining the microbes or first dissolved in water and then blended with the
microbes. In each instance, sufficient agitation is required to ensure uniform
dispersal of both the microbes and the polymer in the mixture. The polymer
concentration in the final solution is selected according to the solubility and
viscosity of the polymer in water and the drying process to be used, but it is
typically between 10 to 30% by weight. The amount of microbes in the final
solution is determined empirically based on the size and shape of the microbe and
the concentration of the polymer. For example, a liquid spore concentrate
containing 15% solids and 1.5 x 10'~ cfus/ml (the spores are sphere shape and 1.5-4
microns in diameter) can be prepared to give a final solution cont~ining 30% (w/w)
of the spore concentrate, 20% (w/w) of skim milk and 5% (w/w) of sucrose for
both spray drying and freeze drying.
The mixture cont~ining polymer and microbes can then be spray dried,
freeze dried or simply air dried to remove unbounded water. Depending on the
polymer used, the drying process can also affect the solubility of the finished
products. In general, freeze drying produces more soluble products than spray
drying, and spray drying produces more soluble products than air drying. PVP is a
suitable polymer for freeze drying, spray drying and air drying on a flat surface.
Skim milk and other protein materials are suitable for freeze drying and spray
drying but not air drying on a flat surface. Polyethylene glycol is more suitable for
CA 022~4431 1998-11-24
freeze drying. The freeze drying on a flat surface is carried out without forming
freezing beads.
The moisture content of the finished product is preferable at the range 1 to
6% by weight or water activity less than 0.4 for better shelf life at ambient
S temperatures.
The finished product is preferably packaged in sealed foil packages in a
nitrogen atmosphere. Packaged in this manner, the finished product has retained at
least 80% effectiveness after storage periods of up to one year at ambient
temperatures.
The present invention is further illustrated by the following examples,
without limiting the scope of the invention. It will be apparent to those skilled in
the art that certain changes can be made to this invention without departing from
the concept or scope of the invention as it is set forth herein.
EXAMPLE 1
Conidiospores of the fungus Penicillium bilaii (ATCC No. 20851) produced
by submerged fermentation were concentrated into liquid spore concentrate after
passing through a 300-mesh screen. 50 g of the liquid spore concentrate cont~ining
18% solids was admixed with 50 g of an aqueous solution cont~ining 25% (w/w)
of PVP (molecular weight 10,000). After mixing for 30 min, the solution was
20 spread out as a 3-mm-thick layer on a flat surface and air dried at 22~C for 24 hrs.
In another treatment, 50 g of the liquid spore concentrate was admixed with 50 gof distilled water and dried under the same conditions as the PVP solution. The air
dried products were assayed for cfu recoveries as given in Table 1 and examined
for solubility after reconstitution in 10 volumes of water at room temperature. The
25 PVP matrix dissolved and completely released the spores entrapped within 5 min
with slight agitation. The spores dried without PVP formed irreversible aggregates
and the spores bounded in the aggregates could not be dissociated in water even
after 8 hrs with strong agitation.
. .~_
CA 022~4431 1998-11-24
TABLE I
Treatment cfus before drying* cfus after drying cfu recovery
PVP (12.5%) 2.2 x 10~~/g 1.9 x 10'~/g86.4%
Water 2.8 x 10'~/g 4.6 x 108/g1.6%
5 * Values of cfus before drying given in this table are standardized as number of
cfus per gram of dry product equivalent to facilitate the comparison before and
after drying.
EXAMPLE 2
50 g of liquid spore concentrate of P. bilaii cont~inin~ about 17% solids
was admixed either with 150 g of aqueous solution cont~ining 17% (w/w) PVP
(molecular weight 10,000), or with 150 g of 14% (w/w) of skim milk solution, or
with 150 g of distilled water. After mixing for 30 min, each of the three solutions
was spray dried using a Buchi 190 lab scale spray dryer set at inlet temperature130~C and outlet temperature 60~C. The spray dried products were assayed for cfu15 recoveries as given in Table 2 and examined for solubility after reconstitution in
10 volumes of water at room temperature. Both PVP and skim milk matrixes
dissolved and the spores dispersed in water within 5 min with slight agitation. The
spores dried without polymers formed irreversible aggregates.
TABLE 2
20 Treatment cfus before drying* cfus after drying cfu recovery
PVP (12.5%) 9.5 x 109/g 6.3 x 109/g66.3%
Skim milk (10%) 1.1 x 10~~/g 5.8 x 109/g 52.7%
Water 1.1 x 10~~/g 1.8 x 109/g 16.3%
* Values of cfus before drying given in this table are standardized as number of25 cfus per gram of dry product equivalent to facilitate the comparison before and
after drying.
CA 022~4431 1998-11-24
EXAMPLE 3
30 kg of P. bilaii liquid spore concentrate cont~ining about 18% solids was
admixed with 70 kg of aqueous solution cont~ining 20 kg of skim milk and 5 kg ofsucrose and 2.5 kg of monosodium gh1t~m~te in a 150-L mixing tank with a 1/2 hp
air-motor mixer for 1 hr. The mixture was then dispensed as a 2.5-cm-thick layerin drying trays, frozen at -20~C for 24 hrs and freeze dried in a commercial scale
shelf dryer for 45 hrs. The freeze dried product had a cfu recovery 83.6%, titer1.64 x 10'~ cfus/g of dry product and moisture content 6%. The dry product is
porous in structure and almost all the spores were trapped and unbounded from
10 each other in the matrix as showing in Figures 3A and 3B. Upon reconstitution,
the matrix dissolved and the spores dispersed in cold or room temperature water
within one minute. Figure 4 shows the spores released after the matrix dissolved.
No measurable irreversible aggregation of the spores was found as indicated by
particle size analysis (Figure 5). When liquid spore concentrates of P. bilaii were
15 freeze dried without using the method of the present invention, the cfu recoveries
were consistently below 1% even in the presence of cryoprotectants, mainly due to
the formation of irreversible aggregates of the spores. The spore aggregates could
not be properly analyzed for particle size distribution because of their quick settling
in solution.
The dry product was stored in sealed foil packages in a nitrogen
atmosphere. It retained at least 80% effectiveness after being stored for one year at
room or cold temperature.
EXAMPLE 4
20 g of P. bilaii liquid spore concentrate containing about 20% solids was
25 admixed with 80 g of aqueous solution to give a final solution containing 20%(w/w) skim milk, 5% (w/w) sucrose and 5% (w/w) dimethylsulfoxide. The mixture
was then extruded drop-wise into liquid nitrogen to form frozen granules with
diameter from 4 to 8 mm and subsequently freeze dried in a Dura-Dry II MPTM
tray dryer. The freeze dried granules had a cfu recovery 27% and titer 5.2 x 10930 cfus/g of dry product. Upon reconstitution in water, the matrix dissolved and released spores instantly.
CA 022~4431 1998-11-24
12
EXAMPLE S
Conidiospores of the fungus Trichoderma harzianum (ATCC No. 20671)
produced by submerged fermentation were concentrated into liquid spore
concentrate after passing through a 300-mesh screen. 10 g of the liquid spore
5 concentrate containing 15% solids was admixed either with 10 g of aqueous
solution of 25% (w/w) PVP or with 10 g of distilled water. The mixtures were
then air dried under the conditions described for EXAMPLE 1. The final dry
products were assayed for cfu recoveries as given in Table 3 and examined for
solubility after reconstitution in water at room temperature. The PVP matrix
10 dissolved and released the spores entrapped within 5 min with slight agitation. The
spores dried without PVP formed large irreversible aggregates.
TABLE 3
Treatment cfus before drying* cfus after drying cfu recovery
PVP (12.5%) 6.0x 109/g 4.8 x 109/g 80.0%
Water 6.0 x 109/g 8.0 x 108/g 13.3%
* Values of cfus before drying given in this table are standardized as number ofcfus per gram of dry product equivalent to facilitate the comparison before and
after drying.
EXAMPLE 6
10 g of liquid spore concentrate of T. harzianum conidiospores cont:~ining
about 15% solids was admixed either with 10 g of an aqueous solution cont~ining
20% (w/w) PVP and 2.5% (w/w) sucrose, or with 10 g of 40% (w/w) skim milk
solution cont~ining 5% sucrose, or with 10 g of distilled water. After mixing for
30 min, the mixtures were frozen at -20~C for 24 hrs and freeze dried in a
Dura-Dry II MPTM tray dryer for 20 hrs. The freeze dried product had cfu
recoveries 87.2%, 82.5% and 6.1% for the polymer matrix treatments PVP, skim
milk, and water, respectively. Upon reconstitution in water, the PVP and skim
milk matrixes dissolved and released the spores ~ apped instantly. Particle sizeanalyses showed no measurable irreversible aggregation of the spores entrapped in
..... .~ _ .. ..
CA 022~4431 1998-11-24
the PVP or skim milk matrixes (Figure 6). The freeze dried product without the
polymers, the spores formed large irreversible aggregates which could not be
properly analyzed for particle size distribution.
EXAMPLE 7
Conidiospores of the fungus Beauveria bassinana (ATCC No. 48023)
produced by submerged fermentation were concentrated into liquid spores
concentrate after passing through a 300-mesh screen. lO g of the liquid spore
concentrate containing about 15% solids was admixed either with 10 g of aqueous
solution cont~ining 20% (w/w) PVP or with 10 g of distilled water. The mixtures
10 were then air dried under the conditions described for EXAMPLE 1. The dried
PVP product had a cfu recovery 72%, and the matrix dissolved in water and
released the spores within 5 min with slight agitation. The spores dried withoutPVP formed large irreversible aggregates and had a cfu recovery 19.5%.
EXAMPLE 8
10 g of B. bassinana liquid spore concentrate cont~ining about 15% solids
was admixed either with 10 g of solution cont~ining 20% (w/w) PVP and 2.5%
(w/w) sucrose, or with 10 g of 40% (w/w) skim milk solution cont:~ining 2.5%
sucrose, or with 10 g of distilled water. After mixing for 30 min, the mixtures
were frozen at -20~C for 24 hrs and freeze dried in a Dura-Dry II MPTM tray dryer
20 for 20 hrs. The freeze dried product had cfu recoveries 56.6%, 78.6% and 25.8%
for the polymer matrix treatments PVP, skim milk, and water, respectively. Upon
reconstitution in water, the PVP and skim milk matrixes dissolved and released the
spores entrapped instantly. Particle size analyses indicated no measurable
irreversible aggregation of the spores dried in the skim milk matrix and slight
25 aggregation of the spores dried in the PVP matrix (Figure 7). The spores dried
without the polymers formed large irreversible aggregates which could not be
properly analyzed for particle size distribution.
