U.S. patent application number 13/321708 was filed with the patent office on 2012-05-31 for stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making.
Invention is credited to Elena Artimovich, Brian Carpenter, Roger Drewes, Moti Harel.
Application Number | 20120135017 13/321708 |
Document ID | / |
Family ID | 43223330 |
Filed Date | 2012-05-31 |
United States Patent
Application |
20120135017 |
Kind Code |
A1 |
Harel; Moti ; et
al. |
May 31, 2012 |
STABLE DRY POWDER COMPOSITION COMPRISING BIOLOGICALLY ACTIVE
MICROORGANISMS AND/OR BIOACTIVE MATERIALS AND METHODS OF MAKING
Abstract
The present invention relates to embedding live or dead
microorganisms and/or bioactive materials in a protective dry
formulation matrix, wherein the formulation includes the bioactive
microorganism or material, a formulation stabilizer agent, and a
protective agent. The formulation is prepared by dispersing all the
solid components in a solution, with or without a vacuum, and
cooling the solution to a temperature above its freezing
temperature. The methods include a primary drying step of the
formulation at a desired temperature and time period, and an
accelerated secondary drying step under maximum vacuum and elevated
temperature, to achieve a final desirable water activity of the dry
material.
Inventors: |
Harel; Moti; (Pikesville,
MD) ; Drewes; Roger; (Hockessin, DE) ;
Carpenter; Brian; (Baltimore, MD) ; Artimovich;
Elena; (Columbia, MD) ; Drewes; Roger;
(Hockessin, DE) ; Carpenter; Brian; (Baltimore,
MD) ; Artimovich; Elena; (Columbia, MD) |
Family ID: |
43223330 |
Appl. No.: |
13/321708 |
Filed: |
May 26, 2010 |
PCT Filed: |
May 26, 2010 |
PCT NO: |
PCT/US2010/036098 |
371 Date: |
February 6, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61181248 |
May 26, 2009 |
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61223295 |
Jul 6, 2009 |
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Current U.S.
Class: |
424/184.1 ;
424/780; 424/93.1; 424/93.4; 435/188; 435/235.1; 435/252.1;
435/255.7; 435/257.1; 435/260; 530/300; 530/350; 530/399 |
Current CPC
Class: |
A23K 50/42 20160501;
A23L 33/40 20160801; A61K 47/46 20130101; A61K 47/42 20130101; A61K
47/38 20130101; A23L 33/135 20160801; Y02A 40/818 20180101; C12N
1/04 20130101; A23K 20/174 20160501; A61K 9/143 20130101; C07K
14/001 20130101; A61P 31/00 20180101; A61K 9/06 20130101; C12N
11/04 20130101; A23L 33/15 20160801; C12N 1/20 20130101; A23K 10/18
20160501; A61K 47/36 20130101; A61K 9/146 20130101; A61P 37/02
20180101; A23K 40/30 20160501; A23K 50/80 20160501; A23L 29/06
20160801; A23K 20/189 20160501; A61K 9/19 20130101 |
Class at
Publication: |
424/184.1 ;
435/260; 435/235.1; 435/255.7; 435/257.1; 530/350; 435/188;
530/300; 530/399; 424/780; 424/93.1; 435/252.1; 424/93.4 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12N 7/00 20060101 C12N007/00; C12N 1/16 20060101
C12N001/16; C12N 1/12 20060101 C12N001/12; C07K 14/00 20060101
C07K014/00; C12N 9/96 20060101 C12N009/96; C07K 2/00 20060101
C07K002/00; C07K 14/575 20060101 C07K014/575; A01N 63/04 20060101
A01N063/04; A01N 63/02 20060101 A01N063/02; A01N 65/03 20090101
A01N065/03; A61K 35/66 20060101 A61K035/66; A61P 31/00 20060101
A61P031/00; C12N 1/20 20060101 C12N001/20; A61K 35/74 20060101
A61K035/74; A61P 37/02 20060101 A61P037/02; C12N 1/04 20060101
C12N001/04 |
Claims
1. A composition comprising (i) a bioactive microorganism or
material as fresh, frozen or dry powder, (ii) at least two
stabilizer agents, and (iii) at least two protective agents,
wherein the composition is suitable for a liquid state drying and
stabilizing the bioactive microorganism or material.
2. The composition of claim 1, wherein total solids range from
about 30 weight percent to about 70 weight percent.
3. The composition of claim 1, wherein the bioactive microorganism
or material is selected from a cell, a microbe, a virus, a culture,
a probiotic, a yeast, an algae, a protein, a recombinant protein,
an enzyme, a peptide, a hormone, a vaccine, a drug, a vitamin, a
mineral, a microbiocide, a fungicide, a herbicide, an insecticide
or a spermicide.
4. The composition according to claim 1, wherein the stabilizer
agent is a polysaccharide and or an oligosaccharide.
5. The composition of claim 4, wherein the polysaccharides is
selected from cellulose acetate phthalate (CAP),
carboxy-methyl-cellulose, pectin, sodium alginate, salts of alginic
acid, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose,
carrageenan, guar gum, gum acacia, xanthan gum, locust bean gum,
chitosan and chitosan derivatives, collagen, polyglycolic acid,
starches and modified starches, cyclodextrins and combinations
thereof.
6. The composition of claim 4, wherein the oligosaccharide is
selected from inulin, maltodextrins, dextrans and combinations
thereof.
7. The composition of claim 4, wherein the stabilizers are present
in an amount ranging from about 1 weight percent to about 20 weight
percent.
8. The composition according to claim 1, wherein the protective
agents are readily soluble in a solution and do not thicken or
polymerize upon contact with water.
9. The composition according to claim 1, wherein the protective
agents are: proteins, such as human and bovine serum albumin, egg
albumen, gelatin, immunoglobulins, isolated soya protein, wheat
protein, skim milk powder, caseinate, whey protein, pea protein and
any protein hydrolysates; carbohydrates including, monosaccharides
(galactose, D-mannose, sorbose, etc.), disaccharides (lactose,
trehalose, sucrose, etc.), cyclodextrins; an amino acid such as
lysine, monosodium glutamate, glycine, alanine, arginine or
histidine, as well as hydrophobic amino acids (tryptophan,
tyrosine, leucine, phenylalanine, etc.); a methylamine such as
betaine; an excipient salt such as magnesium sulfate; a polyol such
as trihydric or higher sugar alcohols, e.g. glycerin, erythritol,
glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene
glycol; polyethylene glycol; pluronics; surfactants; and
combinations thereof.
10. The composition of claim 9, wherein the protective agents are
present in an amount ranging from about 1 weight percent to about
80 weight percent.
11. The composition of claim 1, wherein the total amount of the
protective agents is between 20 and 70 weight percent.
12. A method for preparing a stable dry powder composition of claim
1, such method comprising: (i) combining under vacuum a bioactive
microorganism or material in the form of fresh, frozen or dry
powder, dry stabilizer agents and dry protective agents in an
aqueous solvent; (ii) cooling the mixture of step (i) to a
temperature above its freezing temperature; (iii) primary drying of
the cooled mixture by evaporation, under vacuum, at a temperature
above its freezing temperature; (iv) secondary drying of the
mixture at a temperature of 20.degree. C. or more for a time
sufficient to reduce the water activity of the mixture to Aw-0.3 or
less.
13. The method of claim 12, wherein the mixture is cooled before
drying to a temperature above its freezing temperature.
14. The method of claim 12, wherein the drying of the mixture is
done by evaporation.
15. The method of claim 12, wherein the temperature of the mixture
during the primary drying step is above its freezing
temperature.
16. The method of claim 12, wherein the secondary drying step is
started when the temperature of the mixture is increased by at
least 10.degree. C. above its initial drying temperature.
17. The method of claim 12, wherein the evaporation rate of the
remaining solvent during the secondary drying step is accelerated
by increasing the temperature and/or vacuum pressure.
18. The method of claim 12, wherein the mixture is dried for a time
sufficient to reduce the formulation to a water activity of Aw-0.3
or less.
19. The method of claim 12, wherein the dried mixture is cut,
crushed, milled or respectively pulverized into a free flowing
powder.
20. The method of claim 19, wherein particle size of the dried
mixture is less than about 1000 .mu.m.
21. The method of claim 12, further comprising administering the
composition to a mammal as a reconstituted liquid or as a ground
powder and as a food or feed product.
22. The composition of claim 1, wherein the protecting agents are
dissolved in the solution and wherein the composition has a
viscosity from about 10,000 cP to about 450,000 cP.
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. The method of claim 12, wherein the protecting agents are
dissolved in the aqueous solution and the viscosity of the solution
is from about 10,000 cP to about 450,000 cP to form a homogenous
mixture and wherein vacuum pressure of step (iv) is less than 0.2
Torr for a time sufficient to reduce the water activity of the
mixture to Aw-0.3 or less to form a dry mixture.
31. The method according to claim 30, wherein the dried mixture is
cut, crushed, milled or respectively pulverized into a free flowing
powder.
32. The method according to claim 30, wherein particle size of the
dried mixture is less than about 1000 .mu.m.
33. The method according to claim 30, wherein the bioactive
microorganism or material is selected from a cell, a microbe, a
virus, a culture, a probiotic, a yeast, an algae, a protein, a
recombinant protein, an enzyme, a peptide, a hormone, a vaccine, a
drug, a vitamin, a mineral, a microbiocide, a fungicide, a
herbicide, an insecticide or a spermicide.
34. The method according to claim 30, wherein the dry stabilizer
agents comprise a polysaccharide and an oligosaccharide.
35. The method according to claim 34, wherein the polysaccharide is
selected from cellulose acetate phthalate (CAP),
carboxy-methyl-cellulose, pectin, sodium alginate, salts of alginic
acid, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose,
carrageenan, guar gum, gum acacia, xanthan gum, locust bean gum,
chitosan and chitosan derivatives, collagen, polyglycolic acid,
starches and modified starches, cyclodextrins and combinations
thereof.
36. The method according to claim 34, wherein the oligosaccharide
is selected from inulin, maltodextrins, dextrans and combinations
thereof.
37. The method according to claim 30, wherein the protective agents
do not thicken or polymerize upon contact with water.
38. The method according to claim 30, wherein the protective agent
is selected from a group consisting of human serum, bovine serum
albumin, egg albumen, gelatin, immunoglobulins, isolated soya
protein, wheat protein, skim milk powder, caseinate, whey protein,
pea protein, a protein hydrolysate, galactose, D-mannose, sorbose,
lactose, trehalose, sucrose, cyclodextrins, lysine, monosodium
glutamate, glycine, alanine, arginine, histidine, tryptophan,
tyrosine, leucine, phenylalanine, betaine, magnesium sulfate,
glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol,
mannitol; propylene glycol; polyethylene glycol; pluronics;
surfactants; and combinations thereof.
39. The method of claim 30, wherein the bioactive microorganism or
material is added to the aqueous solution after the protecting
agents are dissolved.
40. A method of feeding an animal a bioactive microorganism or
material, the method comprising: administering a composition
according to claim 22.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Nos. 61/181,248 and 61/223,295 filed in the United
States Patent and Trademark Office on May 26, 2009 and Jul. 6,
2009, respectively, the contents of which are hereby incorporated
by reference herein for all purposes.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is in the field of protection of
bioactive microorganism and/or materials in high temperature and
humid conditions. In particular, the invention relates to embedding
live microorganisms and/or bioactive materials in a protective dry
formulation matrix.
[0004] 2. Related Background Art
[0005] Bioactive microorganisms, such as live or dead bacteria and
viruses, or bioactive materials, such as proteins, vitamins,
minerals, hormones and cells are generally unstable when stored
under conditions of high temperature and humidity. For example,
many commercially available probiotic bacteria such as
lactobacillus rhamnosus can loose more than one log of viability in
less than two weeks when stored in ambient atmosphere at room
temperature (approximately 25.degree. C.). A common process to dry
and protect these bioactive microorganisms after harvesting from a
culture vessel (e.g., fermentor) is to drop a concentrated solution
of the living cells into liquid nitrogen then store the frozen
beads in -80.degree. C. refrigeration for later freeze drying or
shipment to other locations. Freeze-drying has been a dominant
method for drying sensitive bioactive material. Other methods, such
as spray drying, supercritical fluid drying, and desiccation are
generally not suitable for sensitive bioactives such as live or
attenuated bacteria and viruses because of the high drying
temperatures used in these methods which result in significant
damage to the microorganism itself. In addition, they may not
sufficiently dry the material to the specific residual moisture
requirements for product stability, and thus an additional drying
step by other means, may be required.
[0006] In freeze-drying, the bioactive microorganism or materials
is commonly mixed in a solution or suspension of protective agents,
frozen, and then dehydrated by sublimation under full vacuum. The
low temperatures of the freeze-drying process decrease the
degradation reactions of the bioactive and minimize the loss of
activity in the final dry form. However, the requirement for
sub-zero temperatures is energy intensive, and the low surface area
to volume ratios of the frozen material necessitates the use of
long drying time (up to several days per batch cycle). The slow
drying of the freeze-drying process also facilitates the formation
of ice crystals that can damage or denature a sensitive bioactive.
For this reason, bioactive microorganism or materials such as
viruses, bacteria, and cells that possess a cell wall or lipid
membrane, pose significant challenges to the freeze-drying
process.
[0007] One option to reduce the formation of an ice crystal
structure is to add cryoprotective agents to the bioactive
solution. Such protective agents are highly soluble chemicals that
are added to a formulation to protect cell membranes and
intracellular proteins during freezing and to enhance stability
during storage. Common stabilizers for live bacteria and viruses
include sugars such as sucrose, glycerol, or sorbitol, at high
concentrations with the cellular material or bioactive (Morgan et
al., 2006; Capela et al., 2006). However, such protective agents
may not penetrate adequately into the cell to protect active
components within the intracellular volume. Therefore, a
significant challenge remains to develop an optimal drying process
and formulation that minimizes drying losses while achieving
adequate storage stability of the dried material.
[0008] Some of the problems associated with the freeze-drying have
been resolved by using a combination of certain formulations and
vacuum drying in a liquid state. Annear (Annear 1962) developed a
formulation containing bacteria in a solution of sugars and amino
acids and a vacuum drying process that involves boiling and foam
formation. Roser et al. (U.S. Pat. No. 6,964,771) disclosed a
similar concept of drying by foam formation that includes a liquid
concentration step followed by boiling and foaming the concentrated
solution (syrup) under vacuum. To mitigate the oxidation and
denaturation damage that can occur during the boiling step,
Bronshtein (U.S. Pat. Nos. 5,766,520, 7,153,472) introduced an
improved protective formula containing carbohydrates and
surfactants. The drying of the protective solution also involved a
stepwise process of concentration under a moderate vacuum before
application of a strong vacuum to cause frothy boiling of the
remaining water to form dry stable foam. In an attempt to eliminate
the boiling step, Busson and Schroeder (U.S. Pat. No. 6,534,087)
have proposed a drying process in a liquid state formulation for
insensitive bioactives using a vacuum oven without boiling, by
applying very mild vacuum pressure above 30 Torr. After achieving a
certain level of drying without boiling the material, heat was
applied at above 20.degree. C. and dried material was harvested
after only a few hours.
[0009] This type of drying process, in which the bioactive solution
is maintained in a liquid state during the entire drying process,
has the advantage of faster drying due to convection of the liquid
during boiling and the increased surface area presented by the
foaming surfaces. However, boiling and foaming require input of a
significant amount of heat to provide the necessary eruption of the
solution. Such a drying process is not well adapted to drying of
sensitive biologicals, such as viable viruses, cells or bacteria
because the applied heat accelerates enzymatic processes (e.g.,
proteolysis), and chemical processes (e.g., oxidation and free
radical attacks), which can destroy the activity or viability of
the biological material.
[0010] The drying process described above is also limited in its
ability to be scaled to a large industrial process. The avoidance
of freezing requires the process to be conducted at lower vacuum
level (>7 Torr) than in conventional freeze drying or spray
freeze drying process cycles. The most significant disadvantage of
the above processes is the inability to control and limit the
expansion of the foam within the vessel, tray or vial. The
uncontrollable eruption and often-excessive foam formation makes it
practically impossible to develop an industrial scale process. The
eruption and foaming nature of the boiling step results in a
portion of material being splattered on the walls of the vessel and
into the drying chamber. To soften the eruption during boiling,
Bronshtein (U.S. Pat. Nos. 6,884,866, 6,306,345) has proposed
special chambers and a controlled temperature/pressure application
protocol that reduces overheating to an acceptable level. Another
approach to contain the eruption and excessive foaming is described
in US. Pat. App. No.: 2008/0229609, in which the bioactive solution
is enclosed in a container or a bag covered with breathable
membranes. Once again, these protocols are difficult to implement
in industrial level and they are difficult to reliably replicate
with different formulations.
[0011] A need remains for a suitable protective formulation that
can be dried in a liquid state and an industrially scaleable method
to dry bioactive microorganisms such as live or dead viruses,
bacteria and cells, particularly at temperatures above freezing.
There is a need particularly for a cost effective scaleable drying
process that is also suitable for applications outside the
pharmaceutical industry such as food and agriculture industries.
Protective formulations and mild drying processes are required to
provide adequate drying without exposure to high temperatures. A
composition is needed that can protect such biologicals in storage
under high temperature and humid conditions. The present invention,
as described below, provides a solution to all of these
challenges.
SUMMARY OF THE INVENTION
[0012] The present invention includes compositions and methods for
preserving bioactive materials, such as peptides, proteins,
hormones, vitamins, minerals, drugs, microbiocides, fungicides,
herbicides, insecticides, spermicides, nucleic acids, antibodies,
vaccines, and/or bioactive microorganism such as bacteria
(probiotic or otherwise), viruses and/or cell suspensions, in
storage. The drying methods provide a process of controllable
expansion of a formulation comprising the bioactive microorganism
or material, a formulation stabilizer agent, and a protective
agent. The formulation is prepared by dispersing all the solid
components in a solution, with or without a vacuum. The solution is
cooled to a temperature above its freezing temperature and dried
under vacuum into a dry composition, which exhibits an unexpectedly
high stability. The methods include a primary drying step of the
formulation at a desired temperature and time period, and an
accelerated secondary drying step under maximum vacuum and elevated
temperature, to achieve a final desirable water activity of the dry
material.
[0013] In one embodiment, the formulation comprises sufficient
amounts of formulation stabilizer agents, in which the
microorganisms are embedded. Examples of a suitable formulation
stabilizer agent include, but are not limited to, cellulose acetate
phthalate (CAP), carboxy-methyl-cellulose, pectin, sodium alginate,
salts of alginic acid, hydroxyl propyl methyl cellulose (HPMC),
methyl cellulose, carrageenan, guar gum, gum acacia, xanthan gum,
locust bean gum, chitosan and chitosan derivatives, collagen,
polyglycolic acid, starches and modified starches, cyclodextrins
and oligosaccharides (inulin, maltodextrins, dextrans, etc.); and
combinations thereof.
[0014] In one particular embodiment, the preferred formulation
stabilizer agent is sodium alginate. Preferably, the formulation
comprises, in percent by weight of total dry matter, 0.1-10%,
preferably 1-6%, more preferably 2-4% of formulation stabilizer
agent. In an additional embodiment, the formulation stabilizer
comprises a mixture of sodium alginate and oligosaccharides in a
weight ratio of 1:1-10, more preferably 1:1-5 of sodium
alginate/oligosaccharides. In yet another embodiment of the present
invention, the formulation stabilizer is cross-linked with divalent
metals ions to form a firm hydrogel.
[0015] In another embodiment, the formulation comprises significant
amounts of protecting agents, in which the microorganisms are
embedded. Examples of a suitable protecting agent include but not
limited to proteins such as human and bovine serum albumin, egg
albumen, gelatin, immunoglobulin, isolated soya protein, wheat
protein, skim milk powder, caseinate, whey protein and any protein
hydrolysates; carbohydrates including monosaccharides (e.g.,
galactose, D-mannose, sorbose, etc.), disaccharides (e.g., lactose,
trehalose, sucrose, etc.), an amino acid such as lysine, monosodium
glutamate, glycine, alanine, arginine or histidine, as well as
hydrophobic amino acids (tryptophan, tyrosine, leucine,
phenylalanine, etc.); a methylamine such as betaine; an excipient
salt such as magnesium sulfate; a polyol such as trihydric or
higher sugar alcohols, (e.g. glycerin, erythritol, glycerol,
arabitol, xylitol, sorbitol, and mannitol); propylene glycol;
polyethylene glycol; pluronics; surfactants; and combinations
thereof.
[0016] In one preferred embodiment, the protecting agent comprises
a mixture of a disaccharide, a protein, and a protein hydrolysate.
In a particular embodiment, the preferred protecting agent is a
mixture of trehalose, soy protein isolate or whey protein and their
hydrolysates. Preferably, the formulation comprises, in percent by
weight of total dry matter, 10-90%, of trehalose, 0.1-30% soy
protein isolate or whey proteins and 0.1-30% soy or whey protein
hydrolysate. Preferably 20-80% of trehalose, 0.1-20% soy protein
isolate or whey proteins and 1-20% soy or whey protein hydrolysate,
more preferably 40-80% of trehalose, 0.1-20% soy protein isolate or
whey proteins and 1-20% soy or whey protein hydrolysate.
[0017] The method of the invention typically includes blending with
or without a vacuum, concentrated solution or dry powder of
bioactive microorganism (e.g., live or dead vaccines, bacteria,
algae, viruses and/or cell suspensions) or a bioactive material
(e.g., peptides, proteins, hormones, vitamins, minerals, drugs,
microbiocides, fungicides, herbicides, insecticides, spermicides,
nucleic acids, antibodies, vaccines), a stabilizer agent, and a
protective agent into a homogeneous formulation, cooling the
formulation to a temperature above its freezing temperature, and
drying under vacuum at a shelf temperature above 20.degree. C.
According to the invention, the drying process can involve a
primary vacuum drying at a shelf temperature of 20.degree. C. or
above, followed by an accelerating secondary drying of the
formulation under maximum vacuum and elevated temperature for a
time sufficient to reduce the water activity of the dried
formulation to 0.3 Aw or less.
[0018] In one embodiment of the mixing method the bioactive
microorganism or material is in a dry stabilized form and is
further dry blended with the dry stabilizer agents and protective
agents. This dry blend is then added to water and mixed under the
appropriate vacuum and agitation to give a homogeneous slurry of
the desired density.
[0019] In another embodiment of the mixing method, the bioactive
microorganism or material is in the form of a concentrated solution
or paste. The solution is mixed with all the other formulation
ingredients before adding to water.
[0020] In yet another embodiment of the mixing method, the
bioactive microorganism or material is in the form of dry powder.
The dry powder is mixed with all the other formulation ingredients
before adding to water.
[0021] In another embodiment of the mixing method, the dry
bioactive microorganism or material is mixed with just a portion of
the formulation ingredients, and this mixture is added to the
pre-formed slurry, prepared from the addition of the other
formulation ingredients to water.
[0022] In preferred embodiments of the drying methods, the
bioactive microorganism is mixed under vacuum in a solution
including a formulation stabilizer agent and a protective agent. In
one particular embodiment, the bioactive microorganism comprises
live bacteria (e.g., probiotic bacteria). Examples of suitable
microorganisms include, but are not limited to, yeasts such as
Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis, moulds
such as Aspergillus, Rhizopus, Mucor, Penicillium and Torulopsis
and bacteria such as the genera Bifidobacterium, Clostridium,
Fusobacterium, Melissococcus, Propionibacterium, Streptococcus,
Enterococcus, Lactococcus, Kocuriaw, Staphylococcus,
Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc,
Weissella, Aerococcus, Oenococcus and Lactobacillus. Specific
examples of suitable probiotic microorganisms would be represented
by the following species and include all culture biotypes within
those species: Aspergillus niger, A. oryzae, Bacillus coagulans, B.
lentus, B. licheniformis, B. mesentericus, B. pumilus, B. subtilis,
B. natto, Bacteroides amylophilus, Bac. capillosus, Bac.
ruminocola, Bac. suis, Bifidobacterium adolescentis, B. animalis,
B. breve, B. bifidum, B. infantis, B. lactis, B. longum, B.
pseudolongum, B. thermophilum, Candida pintolepesii, Clostridium
butyricum, Enterococcus cremoris, E. diacetylactis, E faecium, E.
intermedius, E. lactis, E. muntdi, E. thermophilus, Escherichia
coli, Kluyveromyces fragilis, Lactobacillus acidophilus, L.
alimentarius, L. amylovorus, L. crispatus, L. brevis, L. case 4 L.
curvatus, L. cellobiosus, L. delbrueckii ss. bulgaricus, L
farciminis, L. fermentum, L. gasseri, L. helveticus, L. lactis, L.
plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei, L.
salivarius, Leuconostoc mesenteroides, P. cereviseae (damnosus),
Pediococcus acidilactici, P. pentosaceus, Propionibacterium
freudenreichii, Prop. shermanii, Saccharomyces cereviseae,
Staphylococcus carnosus, Staph. xylosus, Streptococcus infantarius,
Strep. salivarius ss. thermophilus, Strep. Thermophilus and Strep.
lactis.
[0023] In preferred methods, the formulation is mixed under vacuum
at room temperature (e.g., from 20.degree. C. to 30.degree. C.).
After mixing to homogeneity, the formulation is then cooled to a
temperature above the freezing temperature of the formulation.
Typically, the formulation is cooled to between -10.degree. C. to
+10.degree. C., more preferably the formulation is cooled to
between -5.degree. C. and +5.degree. C. In a preferred embodiment,
the cooled formulation is then transferred to a drying chamber
where heating is applied (20.degree. C. or more) while controlling
an initial vacuum pressure at a level to maintain the original
pre-cooling temperature. Typically, the desirable vacuum pressure
is below 7 Torr but no less than 3 Torr. Under these preferred
conditions a controlled expansion of the formulation and subsequent
faster primary drying of the formulation is achieved. To accelerate
the secondary drying, a maximum vacuum pressure is applied and heat
supply temperature may be further elevated to from 30.degree. C. to
60.degree. C. To maximize the stability of the final product the
formulation is preferably dried for a time sufficient to reduce the
water activity of the formulation to Aw=0.3 or less. In a preferred
embodiment of the invention, the secondary drying comprises removal
of water at a pressure of less than 1 Torr, and in an especially
preferred embodiment to less than 0.2 Torr.
[0024] The wet formulation can be in the form of viscous slurry or
hydrogel particles ranging from 0.05 to 10 mm. The dried
formulation can be used directly as a flake, or ground into a
powder with an average particle size from about 10 .mu.m to about
1000 .mu.m. The formulation can be administrated directly to an
animal, including human, as a concentrated powder, as a
reconstituted liquid, (e.g., beverage), or it can be incorporated
either in flake or powder form into an existing food or feed
product.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the stability trend of the probiotic bacteria,
L. rhamnosus, which was subjected to storage at 40.degree. C. and
33% relative humidity.
[0026] FIG. 2 shows the process temperatures and cumulative
viability loss for a formulation process ending with Aw of 0.28
secondary drying step.
[0027] FIG. 3 shows the effect of different formulation stabilizers
on storage stability.
[0028] FIG. 4 shows the effect of alginate viscosity on the
formulation expansion under vacuum.
[0029] FIG. 5 shows the effect of different combinations of
stabilizer agents on bacteria viability.
[0030] FIG. 6 shows the effect of the formulation density on
expansion rate under vacuum.
[0031] FIG. 7 shows the effect of the formulation pre-cooling
temperature on expansion under vacuum.
[0032] FIG. 8 shows the effect of the vacuum pressure on
formulation temperature during primary drying step.
[0033] FIG. 9 shows the effect of the vacuum pressure on drying
rate of the formulation.
[0034] FIG. 10 shows the stability of the probiotic bacteria, L.
acidophilus dried with the formulation and method of the invention
under storage at 37.degree. C. and 33% relative humidity.
[0035] FIG. 11 shows a flow chart of the method of production
stable dry formulation from hydrogel formulation according to the
invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0036] It is to be understood that the terminology used herein is
for the purpose of describing particular embodiments only, and is
not intended to be limiting. As used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "a protein" includes singular
protein or a combination of two or more proteins; reference to
"enzyme", "vitamin", "bacteria", etc., includes singular or
mixtures of several, and the like.
[0037] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0038] "Ambient" room temperatures or conditions are those at any
given time in a given environment. Typically, ambient room
temperature is 22-25.degree. C., ambient atmospheric pressure, and
ambient humidity are readily measured and will vary depending on
the time of year, weather and climactic conditions, altitude,
etc.
[0039] "Degassing" refers to the release of a gas from solution in
a liquid when the partial pressure of the gas is greater than the
applied pressure. This is not boiling, and can often occur at
pressures above a pressure that would boil a solution. For example,
bottled carbonated soft drinks contained a high partial pressure of
CO.sub.2. Removing the bottle cap reduces the partial pressure and
the drink bubbles vigorously (it degasses, but does not boil).
[0040] "Boiling" refers to the rapid phase transition from liquid
to gas that takes place when the temperature of a liquid is above
its boiling temperature. The boiling temperature is the temperature
at which the vapor pressure of a liquid is equal to the applied
pressure. Boiling can be particularly vigorous when heat is added
to a liquid that is already at its boiling point.
[0041] "Water activity" or "Aw" in the context of dried formulation
compositions, refers to the availability of water and represents
the energy status of the water in a system. It is defined as the
vapor pressure of water above a sample divided by that of pure
water at the same temperature. Pure distilled water has a water
activity of exactly one or Aw=1.0.
[0042] "Relative Humidity" or "RH" in the context of storage
stability refers to the amount of water vapor in the air at a given
temperature. Relative humidity is usually less than that required
to saturate the air and expressed in percent of saturation
humidity.
[0043] "Primary drying", with regard to processes described herein,
refers to the drying that takes place from the time of initial
vacuum application to the point where secondary drying starts.
Typically, the bulk of primary drying takes place by extensive
evaporation, while the product temperature remained significantly
lower than the temperatures of the heat source.
[0044] "Secondary drying", with regard to processes described
herein, refers to a drying step that takes place at temperatures
above freezing temperatures of the formulation and near the
temperature of the heat source. In a typical formulation drying
process, a secondary drying step reduces the water activity of the
formulation to an Aw of 0.3 or less.
[0045] "Bioactive microorganism," or "biologically active
microorganism or formulation" refers to live or dead microorganism
preparations, which are in such a form as to permit the biological
activity of the microorganism to be unequivocally effective. "Live
microorganism as dry powder" refers to a bacterial biomass in which
at least 10% W/W is live bacteria. "Dead microorganism as dry
powder" refers to a bacterial biomass in which at least 99.999% is
dead bacteria.
[0046] "Bioactive material", "bioactive composition", "biologically
active material" or "bioactive formulation" refers to preparations,
which are in such a form as to permit the biological activity of
the bioactive ingredients to be unequivocally effective. Such
bioactive materials include but not limited to peptides, proteins,
hormones, vitamins, minerals, drugs, microbiocides, fungicides,
herbicides, insecticides, spermicides, nucleic acids, antibodies,
and vaccines.
[0047] "Stabilizer or Stabilizing agent" refers to compounds or
materials that are added to the formulation to increase the
viscosity of the wet formulation or to form a hydrogel. Examples of
a suitable stabilizer agent include but are not limited to
polysaccharides, such as, cellulose acetate phthalate (CAP),
carboxy-methyl-cellulose, pectin, sodium alginate, salts of alginic
acid, hydroxyl propyl methyl cellulose (HPMC), methyl cellulose,
carrageenan, guar gum, gum acacia, xanthan gum, locust bean gum,
chitosan and chitosan derivatives, collagen, polyglycolic acid,
starches and modified starches, cyclodextrins and oligosaccharides
(inulin, maltodextrins, raffinose, dextrans, etc.) and combinations
thereof.
[0048] "Protecting agent" or "protective agent" or "protectant"
generally refers to compounds or materials that are added to ensure
or increase the stability of the bioactive material during the
drying process and afterwards, or for long-term storage stability
of the dry powder product. Suitable protectants are generally
readily soluble in a solution and do not thicken or polymerize upon
contact with water. Suitable protectants are described below and
include, but are not limited to, proteins such as human and bovine
serum albumin, whey protein, soy protein, caseinate, gelatin,
immunoglobulins, carbohydrates including monosaccharides
(galactose, D-mannose, sorbose, etc.), disaccharides (lactose,
trehalose, sucrose, etc.), an amino acid such as monosodium
glutamate, lysine, glycine, alanine, arginine or histidine, as well
as hydrophobic amino acids (tryptophan, tyrosine, leucine,
phenylalanine, etc.); a methylamine such as betaine; an excipient
salt such as magnesium sulfate; a polyol such as trihydric or
higher sugar alcohols (e.g., glycerin, erythritol, glycerol,
arabitol, xylitol, sorbitol, and mannitol); propylene glycol;
polyethylene glycol; Pluronics; surfactants, and combinations
thereof.
[0049] A "stable" formulation or composition is one in which the
bioactive microorganism or material therein essentially retains its
viability, and/or biological activity upon storage. Stability can
be measured at a selected temperature and humidity conditions for a
selected time period. Trend analysis can be used to estimate an
expected shelf life before a material has actually been in storage
for that time period. For live bacteria, for example, stability is
defined as the time it takes to loose 1 log of CFU/g dry
formulation under predefined conditions of temperature, humidity
and time period.
[0050] "Viability" with regard to bacteria, refers to the ability
to form a colony (CFU or Colony Forming Unit) on a nutrient media
appropriate for the growth of the bacteria. Viability, with regard
to viruses, refers to the ability to infect and reproduce in a
suitable host cell, resulting in the formation of a plaque on a
lawn of host cells.
[0051] The compositions and methods of the present invention solves
the problem of providing a cost effective and industrially scalable
drying processes for producing a dry formulation containing
bioactive microorganisms or materials, such as live or dead
vaccines, bacteria, algae viruses and/or cell suspensions,
peptides, proteins, hormones, vitamins, minerals, drugs,
microbiocides, fungicides, herbicides, insecticides, spermicides,
nucleic acids, antibodies, vaccines with a significantly extended
lifetime in the dry state. The invention provides a formulation
comprising a bioactive microorganism or material with a stabilizer
agent and a protecting agent in a solution, cooling said
formulation to a temperature above its freezing temperature, and
stabilizing the formulation by removing the moisture under a
regimen of reduced pressure while supplying heat to the
composition.
[0052] Most of the viability loss of microorganism during drying
processes can be attributed to a combination of freeze-thaw
stresses and ice crystal formation, high osmotic and oxidative
stresses, shear forces and energy release during bubble cavitations
associated with the "boiling" of the solution under low drying
pressure and high temperature. The present invention provides a
formulation and an industrially scalable drying process that
minimizes losses during the drying and protects the bioactive
microorganism under harsh storage conditions thereafter.
COMPOSITIONS OF THE INVENTION
[0053] The present invention includes formulation compositions of a
bioactive microorganism or material, a stabilizer agent and a
protecting agent in a viscous solution. The formulations of the
invention were found to be inherently different in their physical
structure and function from non-viscous or concentrated
formulations that were dried without pre-cooling. For example,
formulations of the prior art were initially "foamed" to facilitate
effective drying. The foaming step generally resulted in an
extensive boiling and eruption of the solution that is an
unavoidable consequence of the vacuum drying in a liquid state and
as a result, only a very low loading capacity of material in a vial
or a vessel can be achieved (see for example U.S. Pat. No.
6,534,087, in which the thickness of the final foamed product is
less than 2 mm). The compositions and drying methods of the present
invention allow only a limited and controlled expansion of the
formulation thereby enabling much higher loading of material per
drying area and, as a result, can be easily scaled up to the
production of large quantities of material.
[0054] Single cell microorganisms have been shown to benefit
particularly from the formulations and drying methods of the
present invention. In one embodiment, the bioactive microorganism
of the invention is probiotic bacteria. The formulation is prepared
according to the compositions and methods of the invention
including obtaining live probiotic bacteria in concentrated
solution, paste, frozen beads or dry powder from. Mixing the
probiotic bacteria under vacuum with a stabilizer agent and a
protecting agent, cooling the viscous formulation to a temperature
above its freezing temperature, applying sufficient vacuum pressure
to maintain that pre-cooling temperature and supplying a heat
source of 20.degree. C. and above to facilitate water removal.
Maintaining the pre-cooled temperature of the formulation can be by
conduction of heat away from the formulation, and/or by loss of
latent heat due to water evaporation. To further accelerate the
drying process a secondary drying step is applied, at higher vacuum
up to 0.1 Torr and at elevated temperature up to 70.degree. C., to
provide a final composition with water activity with an Aw of 0.3
or less. Such a composition can remain stable in storage conditions
of 40.degree. C. and 33% RH for 60 days or more (see FIG. 1). The
specified processes of the invention have shown to result in the
unexpected ability of the cells to retain their viability beyond
that of established drying processes. The initial viability loss
through the entire drying process according to the present
invention was only 0.3 logs (see FIG. 2).
[0055] Formulations for Preparation of Stable Dry Powder
Compositions
[0056] The constituents to be mixed with the preferred
microorganism or material for the preparation of dry powder
compositions according to the invention, includes a stabilizer
agent and protective agent. Such constituents, when mixed with the
preferred bioactive microorganisms or material, can be processed
according to methods of the invention to provide large quantities
of stable dry compositions for storage and administration of said
microorganisms. The formulation stabilizers can include a mixture
of a polysaccharide and an oligosaccharide. The preferred
polysaccharide, particularly for stabilizing live microorganisms,
was alginate. Because it was surprisingly found that alginate is
superior to other polysaccharides such as pectin and gum acacia in
reducing the drying losses of sensitive biologicals such as
probiotics (FIG. 3). It was also preferred because of its hydrogel
forming characteristics with non-toxic metals at mild temperatures.
Alginate was also found to effectively stabilize the formulation
under vacuum, by providing appropriate viscosity to the formulation
and allowing a controlled expansion of the formulation at a
particular viscosity (FIG. 4).
[0057] Combining an oligosaccharide with the alginate was also
found to further contribute to the overall stability of the
formulation. FIG. 5 shows the effect on storage stability of
different combinations of alginate and oligosaccharides. A
combination of alginate and inulin was the preferred combination in
term of its long storage effect on the probiotic bacteria. In one
embodiment of the invention, at least one of the formulation
stabilizer agents is preferably a gum that can form a firm hydrogel
by cross-linking with metal ions.
[0058] Protective agents of the invention can include various
proteins, peptides, sugars, sugar alcohols and amino acids. The
protective agent is preferably one that does not crystallize and/or
destabilize the biologically active material in the formulation at
freezing temperatures (e.g., -20.degree. C.). It can be beneficial
to include two or more different protective agents to inhibit the
formation of crystals and stabilize the dried bioactive material
formulation in storage conditions for long time periods.
[0059] The wet formulations can include a substantial amount of
total solids (constituents minus the solvent, such as water). A
major portion of the total solids can consist of the bioactive
material, the stabilizer agent and the protective agent. For
example, the bioactive material can be present in the formulation
in a concentration ranging from about 2-50 weight percent, the
stabilizer agent from about 1-20 weight percent, and the protective
agent from about 20-80 weight percent. In another example, the
stabilizer agent can be present in the formulation in a
concentration ranging from about 0.5-10 weight percent, and the
protective agent from about 10-40 weight percent. Preferably, the
wet formulation should have solids content between about 5% and
80%; more preferably between about 30% to 60%. The viscosity of
formulations of the invention are typically greater than 1000
centipoises (cP); more preferably, greater than 10,000 cP and less
than 450,000; and most preferably greater than 30,000 cP and less
than 100,000 cP.
[0060] The viscosity of formulations of the invention can be as
high as 450,000 cP, provided that the protective agents are
completely dissolved in the solution. Highly viscous and homogenous
slurries containing substantial amount of total solids can be
achieved at elevated temperature, depending on the thermo and
osmo-sensitivity of the bioactive material. For example, live cells
formulations containing 30-60% of total solids can be mixed at
elevated temperature of about 35-40.degree. C. and the mixing is
carried out until all the protective agents are completely
dissolved.
METHODS OF PREPARING STABLE DRY FORMULATIONS
[0061] Methods for preparing stable dry formulations for the
preservation of bioactive microorganisms include, obtaining a live
culture of a specific microorganism in a concentrated solution,
paste, frozen beads or dry powder from (stabilized or otherwise).
Preparation of a formulation by mixing, under vacuum, the bioactive
microorganism or material with a stabilizer agent and a protecting
agent in a solution, cooling the formulation to a temperature of no
more than 10.degree. C. above its freezing temperature, and drying
the formulation by evaporating the moisture under reduced pressure
while supplying heat to the formulation.
[0062] In one embodiment, for example, a formulation comprising a
bioactive microorganism or material, a formulation stabilizer
agent, and a protecting agent are mixed to homogeneity, under mild
vacuum of about 10-50 Torr, in a solution. FIG. 6 shows the effect
of different densities of the formulation on its expansion under
vacuum. The introduction of air during mixing of the formulation
constituents in a solution results in excessive and un-controllable
foaming even at relatively high vacuum pressure. The mixing under
vacuum step according to the invention addresses this problem by
eliminating the introduction of air or gas into the formulation
solution, thereby eliminating excessive and uncontrolled foaming of
the solution.
[0063] The solution is then cooled down to a temperature above its
freezing point (usually between -5.degree. C. and +5.degree. C.).
FIG. 7 shows the effect of pre-cooling of the formulation solution
on its expansion under vacuum pressure. It was surprisingly and
unexpectedly found that boiling can be effectively eliminated even
under a relatively higher vacuum pressure and formulation expansion
is better controlled when the solution temperature is reduced to no
more than 10.degree. C. above its freezing temperature. As can be
seen from FIG. 7, a vacuum pressure of 3 Torr can be applied
without excessive foaming provided that the formulation is cooled
to +5.degree. C. and preferably to -3.degree. C.
[0064] Once cooled, the formulation is then dried under sufficient
vacuum (e.g., about 3 Torr) to maintain that pre-cooled temperature
during the primary drying step. FIG. 8 shows the effect of the
applied vacuum pressure on the temperature of the formulation
solution. At relatively high vacuum pressure above 8 Torr, the
formulation temperature increased to over 6.degree. C. and will
continued to rapidly increase toward the shelf or chamber
temperature. At the same time, the solution will continue foaming
and further expanding. This embodiment is distinguished from the
prior art discussed above (see for example U.S. Pat. No. 6,534,087,
where the applied vacuum pressure is between 3-7 Torr and even
higher), in which a stronger vacuum pressure is applied (<3
Torr) while controlling the expansion of the formulation. This
process results in a significantly faster drying rate (see FIG. 9)
and enables a high loading capacity of the formulation. In this
embodiment, excessive foaming and boiling is eliminated even under
much lower vacuum pressures because the methods of the invention
provide a) a specific composition with a controlled expansion under
vacuum, b) a method that eliminates the introduction of air into
the formulation during mixing and c) a substantial pre-cooling of
the formulation.
[0065] Typical methods in the prior art involve extensive foaming
and/or splattering and violent boiling that can be damaging to
sensitive biologicals and cause difficulties for industrial scale
up. Additionally, a complete and efficient degassing of viscous
slurries is difficult and may require an extended period of time.
These obstacles were resolved in the present invention by first
carrying the entire mixing process under mild vacuum to eliminate
the introduction of entrained gasses into the formulation in the
first place. Any small amount of soluble gases that may remain in
the formulation is then gently removed allowing the formulation to
moderately expand under low vacuum. The additional pre-cooling step
of the formulation to a temperature above its freezing temperature
provides an added control of the expansion rate and thereby allows
much higher loading capacity per drying area than was afforded
according to the prior art. After the primary drying stage is
complete, the stabilized dry formulation can be held at elevated
secondary drying temperatures (up to 70.degree. C.) and vacuum
pressures of less than 0.2 Torr to complete drying of the
formulation in a very short time.
[0066] Another embodiment of the invention provides methods to
prepare hydrogel formulation compositions for preservation of
bioactive microorganisms or materials. For example, a formulation
containing a probiotic bacteria in a dry powder form, a stabilizer
agent and a protective agent, are mixed in a solution, cross-linked
to a hydrogel by adding metal ions or divalent cations and then
dried under low vacuum and temperature as described above. The
pre-cooled temperature of the formulation can be maintained by
conduction of heat away from the formulation, and/or by loss of
latent heat due to water evaporation.
[0067] In one particular embodiment of the invention, for example,
the formulation includes a concentrated fresh or frozen or dry
culture of live probiotic bacteria in a solution of 1 to 2.5%
sodium alginate (preferably 1.5% sodium alginate), 1% to about 5%
inulin (preferably 2.5% inulin), 20% to 60% trehalose (preferably
40% trehalose) and 3% to 15% casein hydrolysate (preferably 8%
casein hydrolysate). The formulation is mixed under vacuum at a
temperature slightly above the room temperature (typically between
25.degree. C.-37.degree. C.) until all the components are
completely dissolved.
[0068] In one additional embodiment of the invention, all the
ingredients are dissolved in the solution at elevated temperature,
then the slurry is cooled down to a temperature between 0.degree.
C. to -5.degree. C. and a dry powder of live microorganism is mixed
in until all the components are completely dissolved. To facilitate
the mixing of the dry live organism powder and to prevent clumping,
a small amount of trehalose can be added to the dry powder
(typically a mixture containing equal portions of dry powder and
trehalose is sufficient.
[0069] The formulation slurry is spread on trays at loading
capacity of about 200 g/sq ft and trays are placed on shelves in a
freeze drier. The shelf temperature is adjusted to 0 to -5.degree.
C. (preferably -2.degree. C.) and the slurry allowed to cool to
that temperature. Vacuum pressure is then applied at 1 to 5 Torr
(preferably 3 Torr) and shelf temperature increased to 20.degree.
to 45.degree. C. (preferably 30.degree. C.) for conductive heat
transfer. The formulation temperature remained at about the
temperature 0 to -5.degree. C. during the primary evaporation step
to prevent the sample from freezing. Secondary drying step at
maximum vacuum of 0.1 Torr and shelf temperature of 40.degree. C.
is started when product temperature reached about +10.degree. C.
The entire drying process proceeds for about 4 hours at which time
the product is harvested and water activity is at Aw-0.3 or
less.
[0070] In another embodiment of the invention, the loaded trays are
pre-cooled to -2.degree. C. in a cold room then immediately loaded
in a vacuum oven drier for radiant heat transfer. The primary and
secondary drying steps are then applied as described above for
conductive heat transfer.
[0071] Preparing the Formulation
[0072] Formulations of the invention can include fresh, frozen or
dry live microorganisms formulated into a solution or suspension
containing a formulation stabilizer agent and a protective agent.
The formulation stabilizer and/or high concentration of protective
agent can be dissolved into a heated aqueous solution with
agitation before cooling and mixing with the bioactive
microorganisms. The microorganisms, such as cultured virus or
bacterium, can be concentrated and separated from culture media by
centrifugation or filtration, then directly mixed into the
formulation of the present invention, or added with conventional
cryoprotectants dropped into liquid nitrogen and the small frozen
beads stored at -80 C until mixed into the formulation.
Alternatively, the frozen beads can be freeze dried, milled into a
fine powder, packed in air tight bags and stored refrigerated until
mixed in the formulation of the invention. In one embodiment of the
present invention, the totality of the water in the formulation is
provided in the liquid of the concentrated live organism and the
live organism suspension is maintained at a temperature slightly
above room temperature. The dry components of the formulation
stabilizer agent and the protective agent are blended together and
then slowly added to the warm suspension of the live organism. The
formulation suspension is gently agitated under mild vacuum in a
planetary mixer until all components are fully dispersed and
uniform slurry is obtained.
[0073] In another embodiment of the present invention the bioactive
microorganism is in the dry powder form and is premixed dry with
formulation ingredients before the resulting dry mixture is added
to water at a temperature slightly above room temperature.
[0074] The bioactive microorganism or material can provide any
bioactivity, such as enzymatic activity, induction of immune
responses, cellular multiplication, infection, inhibition of cell
growth, stimulation of cell growth, therapeutic effects,
pharmacologic effects, antimicrobial effects, and/or the like. The
bioactive microorganism or material can be nonliving cells or
liposomes useful as vaccines or delivery vehicles for therapeutic
agents. Bioactive microorganism of the invention can be live
viruses and live attenuated viruses and/or the like.
[0075] Formulation stabilizers provide structural stability to the
formulation and/or physical and chemical protective benefits to the
bioactive microorganisms. The stabilizers can provide thickening
viscosity to the formulation and better control over its expansion
properties under vacuum pressure and increased structural strength
to the dried formulation compositions of the invention.
[0076] The protective agents can include a variety of proteins,
protein hydrolysates, sugars, sugar alcohols and amino acids. For
example, sugars such as sucrose or trehalose can physically
surround the bioactive material to promote retention of molecular
structure throughout the drying process and impart structural
rigidity to the amorphous matrix in the dry state. The protective
agent can replace water of hydration lost during drying, to prevent
damage to cell membranes and denaturation of enzymes. Other
functions of the protective agents can include protecting the
bioactive material from exposure to damaging light, oxygen,
oxidative agents and moisture. Most protective agents can be
readily dissolved in a solution in amounts ranging from about 0.1
weight percent to about 60 weight percent.
[0077] Pre-Cooling the Formulation
[0078] Formulations of the invention can be pre-cooled before
applying vacuum pressure of the drying process, to provide benefits
such as a further thickening of the formulation slurry, a better
control over the expansion of formulations under low vacuum
pressure, stabilization of bioactive microorganism or material,
and/or enhancing the penetration of formulation constituents
through cell membranes. Cooling can be applied by any appropriate
technique known in the art. For example, cooling can be by contact
and conduction with cold surfaces, loss of latent heat, and/or the
like. Typically, formulations are held in vessels or spread on
metal trays and place in contact with a controlled temperature
surface or a chamber where they equilibrate to the controlled
temperature. Typically, the formulations of the invention can be
pre-cooled to a temperature above its freezing temperature (e.g.,
between -5.degree. C. and +5.degree. C.).
[0079] Primary Drying of the Formulation
[0080] Typical processes for preservation of bioactive
microorganisms such as, live or attenuated organisms include a
combination of freezing and vacuum conditions that can result in
membrane damage and denaturation of cell constituents. The prior
art teaches the use of higher vacuum pressures (e.g., less than 100
Torr), addition of specific cryoprotective agents, concentrating
steps to obtain thick solutions (syrup), and/or higher initial
temperatures to prevent freezing. The use of formulations and
process parameters of the present invention overcome these
limitations and allow for higher loading capacity per drying area
that significantly improves industrial output.
[0081] The formulation in the present invention is dried by
evaporation. Removal of solvent (moisture) from the gaseous
environment around the formulation can be driven by condensation or
desiccation. Evaporation of solvent from the formulation can
provide accelerated primary drying of the formulation under low
vacuum pressure. The controlled expansion of the formulation
accelerates the primary drying of the formulation by rapid transfer
of solvent out of the formulation. The controlled expansion of the
formulation is achieved by gentle degassing (not boiling) of the
remaining dissolved gases when the drying vacuum is applied. Since
it is desirable not to boil or excessively foam the formulation
because the cavitations and shear forces associated with bubble
formation during boiling and/or the formulation may spill out from
containment or have a negative impact on the bioactive
microorganism.
[0082] As primary drying proceeds, the formulation structure is
stabilized. The heat supplied in the drying chamber compensates for
the loss of latent heat caused by evaporation of solvent and the
formulation temperature is maintained within 10.degree. C. above
its freezing temperature. At some point during the primary drying
process, the rate of evaporation of solvent slows and the
formulation temperature begins to increase due to superfluous
supply of heat in the drying chamber. This point indicates the end
of the primary drying step in this invention. As solvent is driven
out from the formulation, the protective agents in solution become
concentrated and thicker until it stops flowing as a liquid. The
amorphous and/or glassy formulation preserves a stable formulation
structure.
[0083] Secondary Drying
[0084] Secondary drying of the structurally stable formulation
removes the remaining entrapped or bound moisture and provides a
composition that is stable in storage for an extended period of
time at ambient temperatures. Secondary drying involves the
application of elevated temperatures and a strong vacuum for
several hours to days. In preferred embodiments the time period
necessary to complete the secondary drying step is double the time
of the primary drying step. Preferably, the water activity of the
formulation at the end of the secondary drying step is less than an
Aw of 0.3. The drying temperature can range from about room
temperature to about 70.degree. C. A typical secondary drying
process for many bioactive microorganisms can include raising the
temperature from about 30.degree. C. to about 40.degree. C., and
holding from about 30 minutes to about 24 hours (preferably from
about 30 minutes to about 4 hours), to provide a stable dried
formulation composition with water activity of less than an Aw of
0.3. In one particular embodiment of the secondary drying, the
drying temperature is slowly raised from primary drying conditions
at a rate that can further preserve the activity of live
biologicals such as live microorganisms. A strong vacuum can be
provided in the secondary drying process to accelerate the rate of
water removal to lower residual moisture levels. The vacuum during
the secondary drying can be less than 1 Torr and, preferably, less
than about 0.2 Torr.
[0085] The drying methods of the invention result in a biologically
active microorganism or bioactive material that is encased within
an amorphous glassy matrix, thereby preventing the unfolding of
proteins and significantly slowing molecular interactions or
cross-reactivity, due to greatly reduced mobility of the compound
and other molecules within the amorphous glassy composition. As
long as the amorphous solid is at a temperature below its glass
transition temperature and the residual moisture remains relatively
low (i.e., below Aw of 0.3), the labile bioactive microorganism can
remain relatively stable. It should be noted that achieving a
glassy state is not a prerequisite for long term stability as some
bioactive microorganisms or materials may fare better in a more
crystalline state.
[0086] Preparation of Dry Powder
[0087] The dried formulation can be used en bloc, cut into desired
shapes and sizes, or crushed and milled into a free flowing powder
that provides easy downstream processing like wet or dry
agglomeration, granulation, tabletting, compaction, pelletization
or any other kind of delivery process. Processes for crushing,
milling, grinding or pulverizing are well known in the art. For
example, a hammer mill, an impact mill, a jet mill, a pin mill, a
Wiley mill, or similar milling device can be used. The preferred
particle size is less than about 1000 .mu.m and preferably less
than 500 .mu.m.
[0088] The compositions and methods described herein preserve the
biological activity of the encased biologically active
microorganism or bioactive materials. For example, the compositions
are tested for stability by subjecting them at elevated temperature
(e.g., 40.degree. C.) and high humidity (e.g. 33% RH) and measuring
the biological activity of the formulations. As an example for live
probiotic bacteria, results of these studies demonstrate that the
bacteria formulated in these formulations are stable for at least
60 days (see FIG. 1). Stability is defined as time for one log
CFU/g potency loss. Such formulations are stable even when high
concentrations of the biologically active material are used. Thus,
these formulations are advantageous in that they may be shipped and
stored at temperatures at or above room temperature for long
periods of time.
EXAMPLES
[0089] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Preparation of Dry Premixed Formulation
[0090] Several formulation premixes were prepared according to
Table 1. Trehalose was obtained from Cargill Minneapolis, Minn. Soy
protein isolate was obtained from Fearn Natural Foods, Mequon, Wis.
Whey protein Concentrate was obtained from Agri-Mark Inc.,
Middlebury, Vt. Casein hydrolysate was obtained from Marcor,
Carlstadt, N.J., and sodium alginate from ISP Corp., Wayne, N.J.
All ingredients were combined together and uniformly mixed (Table
1).
TABLE-US-00001 TABLE 1 Formulations Premix composition (weight
percent) Protein hydrolysate Constituent Soy premix Whey premix
premix Sodium Alginate 3.0 3.0 3.0 Inulin 5.0 5.0 5.0 Trehalose
75.3 75.3 75.3 Soy protein Isolate 14 -- -- Whey protein -- 14 --
concentrate Casein Hydrolysate 2.7 2.7 16.7
Example 2
Stable Dry Powder Containing Probiotic Bacteria
[0091] Lactobacillus Acidophilus (100 g frozen concentrate from a
lab fermentation harvest) was thawed at 37.degree. C. Protein
hydrolysate premix (100 g, Table 1) was slowly added to the thawed
slurry of probiotic bacteria in a jacketed dual planetary mixer
(DPM, 1 qt, Ross Engineering, Inc. Savannah, Ga.). Mixing was
carried out under mild vacuum (25 Torr) at 40 RPM and 37.degree. C.
for 10 minutes. The homogenous slurry was evenly spread on a tray
at a loading capacity of 200 g/sq ft and the tray placed on a shelf
in a freeze drier (Model 25 SRC, Virtis, Gardiner, N.Y.). Shelf
temperature was set above the freezing temperature of the slurry at
-5.degree. C. to cool, but not to freeze, the slurry. Vacuum
pressure (3 Torr) was applied when the formulation temperature
reached about -1.degree. C. The slurry starts to gently degas when
vacuum reached about 7 Torr. When the vacuum reached 3 Torr, the
shelf temperature was increased to 50.degree. C. The formulation
temperature remained at about -1.degree. C. to about +5.degree. C.
during the first 50 minutes of primary drying step. Once the
formulation temperature increased to +10.degree. C., the secondary
drying step was initiated. Maximum vacuum of 0.1 Torr was applied
while still shelf temperature continued to maintain at 50.degree.
C. Secondary drying step was continued for additional 100 minutes,
at which point the drying process was terminated and the dry
formula removed from the freeze drier. The water activity of the
dry formulation at this point was Aw=0.23 as measured by a
Hygropalm Aw1 instrument (Rotonic Instrument Corp., Huntington,
N.Y.).
[0092] The viability losses during formulation, preparation, and
drying processes are presented in FIG. 9. Viability losses during
formulation preparation were 0.26 logs and 0.34 logs during the
drying process for a total cumulative loss of 0.6 logs.
[0093] FIG. 10 shows the storage stability of the dry formulation
under accelerated storage conditions of 37.degree. C. and 33% RH.
After four weeks at these storage conditions, the viability loss of
the probiotic bacteria stabilized in the formulation of the
invention was only 0.8 logs.
Example 3
Preparation of a Hydrogel Formulation
[0094] Concentrated probiotic slurry was prepared according to
Example 2 but using the whey protein premix of Table 1. To this
slurry, 0.5 g of dibasic calcium phosphate was added, followed by
0.5 g of gluconolactone. The slurry was allowed to harden at room
temperature over the next 2 hours to form a solid hydrogel. The
firm gel was sliced to thin and long threads, using a commercially
available slicer/shredder. The thin threads were loaded on a tray
at a loading capacity of 200 g/sq ft and placed in a freeze drier
for drying as described in Example 2. Four hours after establishing
maximum vacuum of 0.1 Torr, the dried product was taken out of the
freeze drier. The water activity (Aw) of the formulation was 0.05
(Measured by HygroPalm Aw1, Rotonic Huntington, N.Y.). The dry
formulation was further ground to fine powder using standard hammer
milling equipment and sieved through 50-250 micron screens. FIG. 11
present a flow chart of the method of production stable dry
formulation from a hydrogel formulation according to the
invention.
Example 4
Preparation of Probiotic Pet Food
[0095] A commercially available pelleted pet food for dogs is dried
in a convection oven to a water activity of 0.1, and then coated
with the stable probiotic dry formulation prepared as described in
Example 3. The dry pellets are sprayed with about 5% of fat-based
moisture barrier (a mixture of 40% chicken fat, 40% cocoa butter
and 20% beeswax), mixed in a drum tumbler with the dry powder
formulation (usually 0.1-0.5% of total pet food that provides a
dosage of 10.sup.8 CFU/g), and finally sprayed with additional coat
of the fat-based moisture barrier. The total amount of coating is
about 15% (of the pet food). Coating time is about 30 min.
Example 5
Preparation of Fish Feed with Several Probiotic Microorganisms
[0096] Pelleted feed for fish according to the present invention
was prepared with a mixture of several probiotics. A stable dry
probiotic formulation containing a mixture of L, rhamnosus, L,
acidophilus and Bifidobacterium lactis was prepared as described in
Example 2. A commercially available starter feed for salmon
(Zeigler Bros., Gardners, Pa.) was first dried in a convection oven
to a water activity of 0.1, and then coated with the probiotics
formulation in a drum tumbler. The pellets (100 g) were first
sprayed with about 5% by weight of fat-based moisture barrier (a
mixture of 40% fish oil, 40% cocoa butter and 20% beeswax), then
mixed with 1 g of the stable dry probiotic formulation (to attain a
dosage of 10.sup.7 cfu/g feed), and finally sprayed with additional
coat of the fat-based moisture barrier. The total amount of coating
was about 10% of the fish feed.
Example 6
An Infant Formula Containing the Dry Formulation of the Present
Invention
[0097] A stable dry formulation containing Lactobacillus GG (Valio
Corp, Finland) is prepared according to Example 2 followed by a
sieving into two particle size groups (above 50 .mu.m and below 150
.mu.m). An infant formula is prepared by mixing 99 g of Nutramigen
(Mead Johnson; Evansville, Ill.) with 0.1 g of the small size
particles (below 50 .mu.m). The final product contains about
10.sup.8 cfu of Lactobacillus GG per 100 g infant formula.
Example 7
Stable Dry Powder Containing an Enzyme
[0098] A hydrogel formula containing 40 weight percent of Savinase
(Novozymes, Denmark) is prepared by mixing, under mild vacuum, 60 g
of protein hydrolysate formulation premix (Table 1) and 40 g of
savinase in 100 g of water solution. The wet formulation is dried
in a vacuum oven at a drying temperature of 50.degree. C. For
determination of loading and storage stability of the dried
formula: a dry sample is accurately weighed (<100 mg) in a
microcentrifuge tube. 200 .mu.l of dimethyl sulfoxide (DMSO) is
added. The formulation is dissolved in the DMSO buffer by
vortexing. To this sample, 0.8 ml of a solution containing 0.05 N
NaOH, 0.5% SDS and 0.075 M Citric acid (trisodium salt) is added.
The tubes are sonicated for 10 min at 45.degree. C., followed by a
brief centrifugation at 5,000 rpm for 10 min. Aliquots of the clear
DMSO/NaOH/SDS/Citrate solution are taken into wells of a microplate
and analyzed for protein content using the Bradford assay method.
The storage stability of the stable enzyme formulation is
significantly higher than a dry enzyme without the formulation of
the present invention.
Example 8
Stable Dry Powder Containing Vitamin A
[0099] A hydrogel formula containing 50 weight percent of Vitamin A
(BASF Corp., Florham Park, N.J.) is prepared by mixing, under 25
Torr vacuum, 50 g of soy protein formulation premix (Table 1) and
50 g of vitamin A powder in 100 g of water solution. The wet
formulation is pre-cooled to -5.degree. C., then spread on trays at
a loading capacity of 200 g/sq ft and dried in a vacuum oven at an
initial vacuum pressure of 3 Torr and temperature of 70.degree. C.,
followed by a maximum vacuum step of 0.2 Torr at 70.degree. C. once
the formulation temperature reached to 5.degree. C.
Example 9
Preparation of Invasive Species Bait
[0100] Pelleted bait for specifically targeted invasive species
according to the present invention is prepared containing a
pesticide. The whey protein premix of Table 1 is added to 200 gm of
water. To this solution is added 90 gm of rotenone and 0.5 gm of
calcium phosphate dibasic, followed by 0.5 gm of gluconolactone.
The slurry is allowed to harden at room temperature over 2 hours.
The firm gel is sliced to thin and long threads through a
slicer/shredder. The thin threads are loaded on a tray and placed
in a vacuum oven dryer. Drying is stopped after achieving a water
activity of 0.10. The dry formulation is ground to the appropriate
size distribution for the bait size specification for the specific
species targeted.
Example 10
Preparation of a Protected Pesticide in a Water-Soluble
Formulation
[0101] A protected soluble granular formulation of a pesticide that
would otherwise be subject to decomposition by other ingredients in
a formulation during storage is prepared by the process of the
present invention. The soy protein premix of Table 1 is added to
200 g of water. To this solution is added 80 g of a dry formulation
of a sensitive formulated pesticide. The slurry is transferred to a
vacuum oven dryer and dried to a water activity of 0.1. The dry
formulation is milled to the desired size and packaged.
Example 11
Preparation of a Protected Pesticide in a Water Insoluble
Formulation
[0102] A protected insoluble granular formulation of a pesticide
that would otherwise be subject to decomposition by other
ingredients in a formulation during storage is prepared with the
formulation and the method of the present invention. The soy
protein premix of Table 1 is added to 200 g water. To this solution
is added 90 g of a dry formulation of a sensitive pesticide and 0.5
g of calcium phosphate dibasic, followed by 0.55 g of
gluconolactone. The slurry is allowed to harden at room temperature
over 2 hours, and then sliced to thin, long threads through a
slicer/shredder. The thin threads are loaded on trays and dried in
a vacuum oven dryer to reach a water activity of 0.1. The dry
formulation is further milled to the desired size distribution and
packaged.
Example 12
[0103] Ten (10) grams of dry Lactobacillus Rhamnosus GG is mixed
with 100 g of the protein hydrolisate premix of Example 1 (table
1). This dry mixture is slowly added to 100 gm of deionized water
at 35.degree. C. in a jacketed dual planetary mixer, and mixed for
10 minutes at 40 rpm. The homogeneous slurry is evenly spread on a
tray at a loading capacity of 100 gm/sq ft, and the tray is placed
on a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner,
N.Y.). The shelf temperature is set at 5.degree. C. to cool the
slurry. Vacuum is applied to reduce the pressure to 3 Torr, at
which time the shelf temperature is raised to 30.degree. C. After 2
hours the pressure is reduced further to 150 milliTorr with the
shelf temperature still held at 30.degree. C. Drying is continued
for an additional 3 hours at which point the product temperature
has risen to within 2.degree. C. of the shelf temperature. The
dried product is then removed from the freeze dryer and the water
activity of the dry formulation at this point is measured by a
Hygropalm Aw1 instrument. Viability losses during formulation,
preparation and drying processes are measured and recorded. Storage
stability testing of the dry formulation is conducted under
accelerated storage conditions of 32.degree. C. and 20% RH.
[0104] Results for the trial at 30.degree. C., and also for one
repeated at 40 C are shown below
TABLE-US-00002 Water Activity after drying 0.25 0.26 Losses during
drying 0.5 log 0.7 log Losses during storage 0.4 log 0.7 log
Example 13
[0105] Twenty (20) grams of dry Lactobacillus Rhamnosus GG is mixed
with 100 g whey protein premix Example 1. This dry mixture is
slowly added to 100 gm of deionized water at 35.degree. C. in a
jacketed dual planetary mixer, and mixed for 10 minutes at 40 rpm.
The homogeneous slurry is evenly spread on a tray at a loading
capacity of 100 gm/sq ft, and the tray is placed on a shelf in a
freeze dryer (Model 25 SRC, Virtis, Gardiner, N.Y.). The shelf
temperature is set at 5.degree. C. to cool the slurry. Vacuum is
applied to reduce the pressure to 3 Torr, at which time the shelf
temperature is raised to 30.degree. C. After 2 hours the pressure
is reduced further to 150 milliTorr with the shelf temperature
still held at 30.degree. C. Drying is continued for an additional 3
hours at which point the product temperature has risen to within
2.degree. C. of the shelf temperature. The dried product is then
removed from the freeze dryer and the water activity of the dry
formulation at this point is measured by a Hygropalm Aw1
instrument. Viability losses during formulation, preparation and
drying processes are measured and recorded.
[0106] Storage stability testing of the dry formulation is
conducted under accelerated storage conditions of 32.degree. C. and
20% RH.
[0107] Results for the trial at 30.degree. C. and also for one
repeated, but run at 40.degree. C. are shown below:
TABLE-US-00003 Water Activity after drying 0.23 0.26 Losses during
drying 0.6 log 0.7 log Losses during storage 0.8 log 0.7 log
Example 14
[0108] Ten (10) grams of dry Lactobacillus acidophilis are mixed
with 10 gms of trehalose and briefly set aside while 65.3 gm of
trehalose, 3 gm of sodium alginate, 5 gm of inulin and 16.7 gm of
whey hydrolysate are mixed together as a dry powder and slowly
added to 100 gm of deionized water at 35.degree. C. in a jacketed
dual planetary mixer, and mixed for 5 minutes at 40 rpm. To this
slurry is added the Lactobacillus acidophilis and trehalose dry
premix, and the mixing is continued for an additional 5 minutes at
35.degree. C. The homogeneous slurry is evenly spread on a tray at
a loading capacity of 100 gm/sq ft, and the tray is placed on a
shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, N.Y.). The
shelf temperature is set at 5.degree. C. to cool the slurry. Vacuum
is applied to reduce the pressure to 3 Torr, at which time the
shelf temperature is raised to 30.degree. C. After 2 hours the
pressure is reduced further to 150 milliTorr with the shelf
temperature still held at 30.degree. C. Drying is continued for an
additional 3 hours at which point the product temperature has risen
to within 2.degree. C. of the shelf temperature. The dried product
is removed from the freeze dryer and the water activity of the dry
formulation at this point is measured using a Hygropalm Aw1
instrument. Viability losses during formulation, preparation and
drying processes are measured and recorded. Storage stability
testing of the dry formulation is conducted under accelerated
storage conditions of 32.degree. C. and 20% RH. Results for the
trial where the dryer is maintained at 30.degree. C. and compared
to those where the dryer is maintained at 50.degree. C. are shown
in Table below.
TABLE-US-00004 Water Activity after drying 0.23 0.26 Losses during
drying 0.6 log 0.7 log Losses during storage 0.8 log 0.9 log
Example 15
[0109] Ten (10) grams of dry Lactobacillus acidophilis are mixed
with 10 gms of trehalose and briefly set aside while 65.3 gm of
trehalose, 3 gm of sodium alginate, 5 gm of inulin and 16.7 gm of
whey hydrolysate are mixed together as a dry powder and slowly
added to 100 gm of deionized water at 50.degree. C. in a jacketed
dual planetary mixer, and mixed for 5 minutes at 40 rpm. The slurry
is cooled down to 4.degree. C. To this cooled slurry is added the
Lactobacillus acidophilis and trehalose premix, and the mixing is
continued for an additional 5 minutes at 4.degree. C. The
homogeneous slurry is evenly spread on a tray at a loading capacity
of 100 gm/sq ft, and the tray is placed on a shelf in a freeze
dryer (Model 25 SRC, Virtis, Gardiner, N.Y.). The shelf temperature
is set at 5.degree. C. to maintain the temperature of the cool
slurry. Vacuum is applied to reduce the pressure to 3 Torr, at
which time the shelf temperature is raised to 30.degree. C. After 2
hours the pressure is reduced further to 150 milliTorr with the
shelf temperature still held at 30.degree. C. Drying is continued
for an additional 3 hours at which point the product temperature
has risen to within 2.degree. C. of the shelf temperature. The
dried product is removed from the freeze dryer. The water activity
of the dry formulation at this point is Aw=0.23 as measured by a
Hygropalm Aw1 instrument. Viability losses during formulation,
preparation and drying processes total 0.6 logs.
Example 16
[0110] One hundred (100) gram of soy premix is slowly added to 100
gm of deionized water at 35.degree. C. in a jacketed dual planetary
mixer, and mixed for 10 minutes at 40 rpm. Ten (10) grams of dry
Bifidobacterium lactis Bb-12 is added slowly with mixing at 20 rpm,
and the slurry mixed for an additional 5 minutes. The homogeneous
slurry is evenly spread on a tray at a loading capacity of 100
gm/sq ft, and the tray is placed on a shelf in a freeze dryer
(Model 25 SRC, Virtis, Gardiner, N.Y.). The shelf temperature is
set at 5.degree. C. to cool the slurry. Vacuum is applied to reduce
the pressure to 3 Torr, at which time the shelf temperature is
raised to 30.degree. C. After 2 hours the pressure is reduced
further to 150 milliTorr with the shelf temperature still held at
30.degree. C. Drying is continued for an additional 3 hours at
which point the product temperature has risen to within 2.degree.
C. of the shelf temperature. The dried product is removed from the
freeze dryer and the water activity of the dry formulation at this
point is Aw=0.26 as measured by a Hygropalm Aw1 instrument.
Viability losses during formulation, preparation and drying
processes total 0.7 logs.
[0111] Storage stability testing of the dry formulation under
accelerated storage conditions of 32.degree. C. and 20% RH show a
viability loss of the stabilized probiotic bacteria in the
formulation of the invention to be only 0.7 logs after four
weeks.
Example 17
[0112] The same parameters as Example #1, except the mixing done in
the Ross mixer is under 25 inches of vacuum to give a slurry
density of 1.2 gm/cc. The water activity of the dry formulation at
this point is Aw=0.26 as measured by a Hygropalm Aw1 instrument.
Viability losses during formulation, preparation and drying
processes total 0.5 logs.
[0113] Storage stability testing of the dry formulation under
accelerated storage conditions of 32.degree. C. and 20% RH show a
viability loss of the stabilized probiotic bacteria in the
formulation of the invention to be only 0.7 logs after four
weeks.
Example 18
[0114] One hundred (100) grams of a fresh liquid concentrate of LGG
bacteria (containing 10% solids and the rest water) is added to a
jacketed dual planetary mixer and warmed to 35.degree. C. To this
is slowly added 100 g of whey premix (Table 1). The resulting
slurry is mixed for 10 minutes at 40 rpm. The homogeneous slurry is
evenly spread on a tray at a loading capacity of 100 gm/sq ft, and
the tray is placed on a shelf in a freeze dryer (Model 25 SRC,
Virtis, Gardiner, N.Y.). The shelf temperature is set at 5.degree.
C. to cool the slurry. Vacuum is applied to reduce the pressure to
3 Torr, at which time the shelf temperature was raised to
30.degree. C. After 2 hours the pressure is reduced further to 150
milliTorr with the shelf temperature still held at 30.degree. C.
Drying is continued for an additional 3 hours at which point the
product temperature has risen to within 2.degree. C. of the shelf
temperature. The dried product is removed from the freeze dryer.
The water activity of the dry formulation at this point is Aw=0.25
as measured by a Hygropalm Aw1 instrument. Viability losses during
formulation, preparation and drying processes total 0.5 logs.
Storage stability testing of the dry formulation under accelerated
storage conditions of 32.degree. C. and 20% RH show a viability
loss of the stabilized probiotic bacteria in the formulation of the
invention to be only 0.4 logs after four weeks.
Example 19
[0115] Lactobacillus Rhamnosus GG (LGG) One hundred (100) grams of
unthawed, frozen concentrate and 100 g of protein hydrolysate
premix were added to a jacketed dual planetary mixer (DPM, 1 pt,
Ross Engineering, Inc., Savannah, Ga.). This process can also be
done by thawing the frozen concentrate first. Mixing was carried
out at 40 RPM and 37.degree. C. for 10 minutes. The homogeneous
slurry was measured for viscosity (Brookfield viscometer, Model #
LVDVE115, Brookfield Engineering Laboratories, Inc.), and then
evenly spread on a tray at a loading capacity of 100 g/sq ft.
Viscosity Parameters for high viscosity ranges were: 300 g sample
in a 400 mL Pyrex beaker, 33-37 C, Spindle #64, 1.0 RPM speed,
operated without a guard-leg. The tray was then loaded into a
-4.degree. C. refrigerator for cooling for 30 min. After cooling,
the drying began using a freeze drier (Model 25 SRC, Virtis,
Gardiner, N.Y.) with a shelf temperature set at 30.degree. C.
throughout, and 2800 mTorr of pressure for at least 2.5 hours.
After at least 2.5 hours, the pressure was decreased to 100 mTorr
for at least another 2.5 hours. This experiment was repeated with
two different batches of LGG fermentate, and included washing of
one batch with 3% DMV and reconstitution with de-ionized water
prior to adding the hydrolysate premix.
TABLE-US-00005 CFU/g of final Losses during Viscosity of Sample
product drying slurry (cP) LGG Batch # 1 2.20 .times. 10.sup.+10
0.61 410,000 LGG Batch # 2 7.00 .times. 10.sup.+10 0.48 N/A Washed
LGG Batch # 2 1.38 .times. 10.sup.+11 0.21 319,000
[0116] Viscosity Parameters for medium viscosity ranges were: 300 g
sample in a 400 mL Pyrex beaker, 33-37.degree. C., Spindle #64, 5.0
RPM speed, operated without a guard-leg.
TABLE-US-00006 CFU/g of final Losses during Viscosity of Sample
product drying slurry (cP) LGG Batch # 4 1.39 .times. 10.sup.+11
0.18 58,100 Washed LGG Batch # 4 1.31 .times. 10.sup.+11 0.23
36,200
Example 20
[0117] Lactobacillus Rhamnosus GG (LGG) One hundred (100) grams of
frozen concentrate was thawed at 37.degree. C. and added to a
jacketed dual planetary mixer (DPM, 1 pt, Ross Engineering, Inc.,
Savannah, Ga.). To it, 100 g of protein hydrolysate premix was
added. Unthawed frozen concentrate may also be used. Mixing was
carried out at 40 RPM and 37.degree. C. for 10 minutes, and then
the slurry was evenly spread onto trays at a loading capacity of
100 g/sq ft. The trays were then loaded into a -4.degree. C.
refrigerator for cooling for 30 min. After cooling, the drying
begins using a freeze drier (Model 25 SRC, Virtis, Gardiner, N.Y.)
with a shelf temperature set at 30.degree. C. throughout, and 2800
mTorr of pressure for at least 2.5 hours. After at least 2.5 hours,
the pressure was decreased to 100 mTorr for at least another 2.5
hours. This same process was applied to 10 g of dried (powdered)
LGG material, which was mixed into 100 g of protein hydrolysate.
This dry mixture was then slowly added to 90 g of de-ionized water
in the jacketed dual planetary mixer.
TABLE-US-00007 Sample Losses during drying (logs) Dry LGG/MM final
product 1.26 Frozen LGG concentrate/MM final product 1.46
Example 21
Stable Dry Powder Containing Enzyme
[0118] Forty (40) gram of proteolitic enzyme (Novozymes, Denmark)
in the form of dry powder is mixed with 60 g of soy premix (Table
1). This dry mixture is slowly added to 100 g of deionized water at
35.degree. C. in a jacketed dual planetary mixer, and mixed for 10
minutes at 40 rpm. The homogeneous slurry is evenly spread on a
tray at a loading capacity of 100 gm/sq ft, and the tray placed on
a shelf in a freeze dryer (Model 25 SRC, Virtis, Gardiner, N.Y.).
The shelf temperature is set at 5.degree. C. to cool the slurry.
Vacuum is applied to reduce the pressure to 3 Torr, at which time
the shelf temperature is raised to 60.degree. C. After 1 hour the
pressure is reduced further to 150 milliTorr with the shelf
temperature still held at 60.degree. C. Drying is continued for an
additional 1 hour at which point the product temperature had risen
to within 2.degree. C. of the shelf temperature. The dried product
is removed from the freeze dryer. For determination of loading and
storage stability of the dried formula: the dry sample is
accurately weighed (<100 mg) in a microcentrifuge tube and 200
.mu.g of dimethyl sulfoxide (DMSO) is added. The formulation is
dissolved in the DMSO buffer by vortexing. To this sample, 0.8 ml
of a solution containing 0.05N NaOH, 0.5% SDS and 0.075M Citric
acid (trisodium salt) is added. The tubes are sonicated for 10 min
at 45.degree. C., followed by a brief centrifugation at 5,000 rpm
for 10 min. Aliquots of the clear DMSO/NaOH/SDS/Citrate solution
are taken into wells of a microplate and analyzed for protein
content using the Bradford assay method. The storage stability of
the stable enzyme formulation is significantly higher than a dry
enzyme without the formulation of the present invention.
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