U.S. patent application number 10/383001 was filed with the patent office on 2004-05-06 for culture medium with yeast or soy bean extract as amino acid source and no protein complexes of animal origin.
This patent application is currently assigned to Chiron S.p.A.. Invention is credited to Giglioli, Antonella, Kontakou, Maria, Olivieri, Roberto, Rappuoli, Rino, Sabbatini, Fabio, Tagliaferri, Lucia.
Application Number | 20040087020 10/383001 |
Document ID | / |
Family ID | 32180225 |
Filed Date | 2004-05-06 |
United States Patent
Application |
20040087020 |
Kind Code |
A1 |
Olivieri, Roberto ; et
al. |
May 6, 2004 |
Culture medium with yeast or soy bean extract as amino acid source
and no protein complexes of animal origin
Abstract
The invention relates to a medium for culturing pathogenic
bacteria to produce an immunogenic factor. The medium comprises
non-animal derived proteinaceous material. The invention also
relates to the use of the medium to cultivate pathogenic bacteria,
obtaining immunogenic factors from the bacteria being cultivated
and preparing vaccines using the immunogenic factors.
Inventors: |
Olivieri, Roberto; (Siena,
IT) ; Sabbatini, Fabio; (Monteroni d'Arbia, IT)
; Kontakou, Maria; (Caserta, IT) ; Tagliaferri,
Lucia; (Siena, IT) ; Giglioli, Antonella;
(Siena, IT) ; Rappuoli, Rino; (Monteriggioni,
IT) |
Correspondence
Address: |
Chiron Corporation
Intellectual Property - R440
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron S.p.A.
Via Fiorentina, 1
Siena
IT
1-53100
|
Family ID: |
32180225 |
Appl. No.: |
10/383001 |
Filed: |
March 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10383001 |
Mar 7, 2003 |
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09424800 |
Feb 24, 2000 |
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09424800 |
Feb 24, 2000 |
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PCT/IB98/00938 |
May 28, 1998 |
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Current U.S.
Class: |
435/404 |
Current CPC
Class: |
A61P 31/04 20180101;
C12N 1/20 20130101; C12R 2001/21 20210501; A61K 39/02 20130101;
C12N 1/205 20210501 |
Class at
Publication: |
435/404 |
International
Class: |
C12N 005/02 |
Foreign Application Data
Date |
Code |
Application Number |
May 28, 1997 |
GB |
9710981.3 |
Mar 30, 1998 |
GB |
9806802.6 |
Claims
1. A medium for cultivating pathogenic bacteria to produce an
immunogenic factor wherein the medium comprises at least 20% by dry
weight of a non-animal derived proteinaceous material, and does not
comprise animal derived proteinaceous material.
2. The medium of claim 1 wherein the non-animal derived
proteinaceous material is a yeast extract and/or a soy bean derived
protein composition.
3. The medium of claim 1 or claim 2 wherein the pathogenic bacteria
are Helicobacter pylori, Haemophilus influenzae, Corynebacterium
diphtheriae, Neisseria meningitidis, Bordetella pertussis or
Clostridium tetani.
4. A culture comprising the medium of any one of the previous
claims and pathogenic bacteria.
5. A process for preparing an immunogenic factor of a pathogenic
bacteria comprising the steps of cultivating the bacteria in the
medium of any one of claims 1 to 3 and optionally purifying the
immunogenic factor from the medium.
6. A process for the production of a vaccine comprising preparing
an immunogenic factor of a pathogenic bacteria by the process of
claim 5, and bringing said factor, optionally toxoided, into
association with a pharmaceutically acceptable carrier.
7. A process for making a medium for cultivating pathogenic
bacteria to produce an immunogenic factor comprising adding
sufficient non-animal derived proteinaceous material to a standard
medium for cultivating bacteria, which does not comprise animal
derived proteinaceous material, so that the medium for cultivating
pathogenic bacteria comprises at least 20% by dry weight of the
non-animal derived proteinaceous material, and does not comprise
animal derived proteinaceous material.
Description
[0001] The present invention relates to a medium for cultivating
pathogenic bacteria. The present invention also relates to the use
of the medium to cultivate pathogenic bacteria, obtaining
immunogenic factors from the bacteria being cultivated and
preparing vaccines using the immunogenic factors.
[0002] Bacterial vaccines are produced by cultivating pathogenic
bacteria in a medium, isolating immunogenic factors and preparing
vaccines based on the isolated immunogenic factors. Such methods
are described in Bacterial Vaccines, 1984, Ed. Rene Germanier
Academic Press and Bacterial Vaccines in Advances in Biotechnology
Processes, Vol. 13, 1990, Ed. A. Mizrahi, Wiley-Liss. In
conventional methods, the pathogenic bacteria are cultivated in
media containing proteinaceous material of animal origin. These
compounds are used in the belief that some growth factors,
essential for the growth of pathogenic bacteria, were only present
in compounds of animal origin such as blood, brain heart infusion,
meat, etc. For instance, C. tetani is grown in media containing a
heart infusion and an enzymatic digest of casein; C. diphtheriae
requires a beef infusion; H. pylori is grown in media containing
peptarnin and tryptone; and Haemophilus influenzae is grown in
media containing proteose peptones. World Health Organisation
report series numbers 800 (1990) and 814 (1991) indicate that to
grow Haemophilus influenza, Corynebacterium diphtheriae,
clostridium tetani and Bordetella pertussis, media comprising
compounds of animal origin are required.
[0003] The requirement for proteinaceous material of animal origin
in the media gives rise to concern over possible contamination of
the media. In particular, concern that the media may be
contaminated with the bovine spongiform encephalopathy (BSE)
causative agent or other infectious and harmful agents, restricts
the usefulness of any factors derived from such cultures,
especially in therapeutic applications.
[0004] It has surprisingly been found that proteinaceous materials
of non-animal origin are able to sustain the growth of pathogenic
bacteria and enable the production of immunogenic factors by the
bacteria.
[0005] In patent application DD 294 502-A, a process for preparing
soya hydrolysates and the use of the soya hydrolysates in culturing
microorganisms for fimbriae is disclosed. The soya hydrolysate is
prepared by culturing soya-flour-containing medium with
streptomyces strains. The soya hydrolysate is used as a carbon
source due to the high percentage of polysaccharides present and
contains less than 20% by dry weight protein.
[0006] The use of proteinaceous material of non-animal origin, such
as proteins from soy beans, cotton seeds, potatoes, etc., as a
media constituent for the cultivation of pathogenic bacteria,
completely removes the risk of animal derived contamination, such
as BSE, being transmitted into humans in any subsequent therapeutic
or prophylactic applications.
[0007] A further advantage associated with the use of vegetable
derived proteinaceous materials is the reduction in cost of
producing the materials and the increased consistency in the
materials (non-animal derived proteinaceous material is more
uniform in its composition than animal derived materials).
[0008] The present invention provides a medium for cultivating
pathogenic bacteria to produce an immunogenic factor wherein the
medium comprises at least 20% by dry weight of a non-animal derived
proteinaceous material, and does not comprise animal derived
proteinaceous material.
[0009] Any standard medium for cultivating bacteria may be used as
the basis for the medium of the present invention, provided the
medium does not contain animal derived proteinaceous material.
[0010] Preferably, the medium of the present invention comprises a
carbon and an energy source, a nitrogen source, essential salts and
optionally a selecting agent, such as an antibiotic, for selecting
the microorganisms to be cultured.
[0011] The medium of the present invention may be a solid or liquid
medium. Examples of standard liquid media which may form the basis
of the medium of the present invention include Brucella Broth
(without tryptone and peptamine), Watson medium (without casamino
acids), Mueller Miller medium (without heart infusion and casein
hydrolysate), CL medium (without casamino acids) and Franz A
medium. Standard solid media can be prepared from any of the liquid
media by the addition of a solidifying agent such as agar.
[0012] The term "cultivating" as used herein means the maintenance
of, and preferably the growth of, bacteria. Bacterial growth is
herein defined as an increase in bacterial biomass.
[0013] The term "pathogenic bacteria" as used herein, means any
bacteria which is involved in the pathogenesis of a disease.
Preferred pathogenic bacteria include Helicobacter pylori,
Haemophilus influenzae, Corynebacterium diphtheriae and Neisseria
meningitidis, Bordetella pertussis and Clostridium tetani.
[0014] The term "immunogenic factor" as used herein, means any
factor which is capable of stimulating the immune system of a human
or animal. Such immunogenic factors include antigenic proteins and
especially virulence factors and fragments thereof Virulence
factors are defined as being associated with the virulence of a
bacteria and includes such factors as the vaculating cytotoxin VacA
produced by Helicobacter pylori. Other virulent factors are
described by Rappuoli, R. et al., European Journal of
Gastroenterology and Hepatology of Helicobacterpylori infection.
Proceedings of an interdisciplinary meeting (Genova, Jun. 18-19,
1993) J. J. Misiewicz, Ed. (CS Current Science), pp S76-S78
(incorporated herein by reference). The immunogenic factor may be
genetically detoxified or treated by a toxoiding process. Methods
for genetically detoxifying and toxoiding immunogenic factors are
well known to those skilled in the art and include those described
by Rappuoli, R., Vaccine, 12, 579-581, (1994) (herein incorporated
by reference).
[0015] The term "proteinaceous material", as used herein, means
proteins and protein degradation products including free amino
acids. Preferably, the proteinaceous material is a protein
hydrolysate.
[0016] Non-animal derived proteinaceous materials as used herein
means proteinaceous materials derived from non-mammalian sources,
such as vegetables, birds, fish, yeasts, fungi, algae and
microorganisms. More preferably, non-animal proteinaceous materials
mean proteinaceous material derived from vegetables, yeasts, algae
and microorganisms. Most preferably, non-animal derived
proteinaceous materials means proteinaceous materials derived from
vegetables such as protein compositions derived from soy beans,
cotton seeds, potatoes, etc.
[0017] Preferred non-animal derived proteinaceous materials include
yeast extracts such as HY YEST (Quest) and soy bean derived protein
compositions such as Hysoy (Quest), Amisoy (Quest), N-Z soy (Quest)
and Soytone (Difco).
[0018] Yeast extracts can be prepared by standard procedures well
known to those skilled in the art. Furthermore, yeast extracts are
commercially available from numerous sources including Sigma and
Quest.
[0019] Soy bean derived protein compositions can be prepared by
enzymatic digestion of soy bean meal or soy isolate using standard
enzymes such as papain. For example, N-Z soy is a soluble protein
composition made by the enzymatic digestion of a soy isolate and
Hysoy is a papaic digest of soy bean meal. Soy bean derived protein
compositions can also be obtained by acid hydrolysis of a soy
isolate. For example, Amisoy is a source of amino acids and
peptides produced by acid hydrolysis of a soy isolate.
[0020] Other non-animal derived proteinaceous materials can be
obtained by either enzymatic digestion or by acid hydrolysis of a
protein containing material of a non-mammalian source.
[0021] The non-animal derived proteinaceous material in the medium
of the present invention may comprise two or more different
non-animal derived proteinaceous materials, such as a mixture of
soy bean derived protein compositions such as Hysoy, Amisoy, N-Z
soy and Soytone.
[0022] Animal derived proteinaceous materials include protein
compositions such as fetal calf serum (FCS), bovine serum albumin
(BSA), proteose peptones, casamino acids, tryptone, peptamin and
casein hydrolyzates.
[0023] Preferably, the medium of the present invention comprises at
least 20% by dry weight of a non-animal derived proteinaceous
material, more preferably, at least 30% by dry weight of a
non-animal derived proteinaceous material and most preferably at
least 50% by dry weight of a non-animal derived proteinaceous
material.
[0024] It has surprisingly been found that the cultivation of
pathogenic bacteria using the medium of the present invention
results in increased growth of the bacteria and an increased yield
of immunogenic factors compared to cultivation of the pathogenic
bacteria in a medium containing animal derived proteinaceous
material.
[0025] The present invention further provides a process for making
a medium for cultivating pathogenic bacteria to produce an
immunogenic factor comprising adding sufficient non-animal derived
proteinaceous material to a standard medium for cultivating
bacteria, which does not comprise animal derived proteinaceous
material, so that the medium for cultivating pathogenic bacteria
comprises at least 20% by dry weight of the non-animal derived
proteinaceous material, and does not comprise animal derived
proteinaceous material.
[0026] Examples of standard liquid media include Brucella Broth
(without tryptone and peptamine) Watson medium (without casamino
acids), Mueller Miller medium (without heart infusion and casein
hydrolysate), CL medium (without casarnino acids) and Franz A
medium. Standard solid media can be prepared from any of the liquid
media by the addition of a solidifying agent such as agar.
[0027] The present invention further provides a culture comprising
the medium of any of the previous claims and pathogenic bacteria.
Preferred pathogenic bacteria include Helicobacter pylori,
Haemophilus influenzae, Corynebacterium diphtheriae and Neiseria
meningitidis, Bordetella pertussis and Clostridium tetani. Most
preferably, the pathogenic bacterium is Helicobacter pylori.
[0028] The present invention also provides a process for preparing
an immunogenic factor of a pathogenic bacteria comprising the steps
of cultivating the bacteria in the medium of the present invention
and optionally purifying the immunogenic factor from the
medium.
[0029] Preferably, the pathogenic bacteria are cultured in the
medium of the present invention for at least 6 hours, more
preferably at least 36 hours, and most preferably at least 72 hours
under suitable conditions for the production of the immunogenic
factor.
[0030] Suitable culture conditions for the production of the
immunogenic factor, including the duration of the culture, will
vary depending on the bacteria being cultured. However, one skilled
in the art can easily determine the culture conditions required for
the production of the immunogenic factor by following standard
protocols, such as those described in the series Methods in
Microbiology, Academic Press Inc., (incorporated herein by
reference) and, if necessary, by performing a number of standard
experiments to determine suitable culture conditions.
[0031] The immunogenic factor can be isolated from the bacterial
culture using a number of standard techniques including those
described by Manetti, R. et al., Infect. Immun., 63, 4476-4480,
(1995), incorporated herein by reference.
[0032] The present invention also provides a process for the
production of a vaccine comprising preparing an immunogenic factor
of a pathogenic bacteria comprising the steps of cultivating the
bacteria in the medium of the present invention, optionally
purifying the immunogenic factor from the medium and bringing said
factor, optionally toxoided, into association with a
pharmaceutically acceptable carrier. Suitable methods for producing
a vaccine are described by Rappuoli, R., New and improved vaccines
against Diphtheria and Tetanus. (1990), 251-268, New Generation of
Vaccines, Ed. G. C. Woodrow, M. M Levine, Marcel Dekker Inc. New
York. (Incorporated herein by reference).
[0033] The vaccines prepared by the process of the present
invention will require the addition of adjuvants when they are
used. Suitable adjuvants are described in Gupta, R. K. et al.,
Vaccine, 13, 1263-1276, (1995).
[0034] The vaccines prepared by the process of the present
invention can be used to vaccinate an individual against a
bacterial infection. Preferred bacterial infections which can be
vaccinated against include type B gastritis, bacterial
meningitidis, diphtheria, tetanus and whooping cough.
[0035] The vaccines prepared by the process of the present
invention may be provided as a pharmaceutical composition
comprising the vaccine of the present invention in admixture with a
pharmaceutically acceptable carrier and adjuvants as mentioned
above.
[0036] The vaccines prepared by the process of the present
invention can be administered by oral or parenteral route,
including intravenous, intramuscular, intraperitoneal, subcuaneous,
transdermal, airway (aerosol), rectal and topical
administration.
[0037] For oral administration, the compounds of the invention will
generally be provided in the form of tablets or capsules or as an
aqueous solution or suspension.
[0038] Tablets for oral use may include the active ingredients (the
vaccine component) mixed with pharmaceutically acceptable
excipients such as inert diluents, disintegrating agents, binding
agents, lubricating agents, sweetening agents, flavouring agents,
colouring agents and preservatives. Suitable inert diluents include
sodium and calcium carbonate, sodium and calcium phosphate, and
lactose, while corn starch and alginic acid are suitable
disintegrating agents. Binding agents may include starch and other
well known agents, while the lubricating agent, if present, will
generally be magnesium stearate, stearic acid or talc. If desired,
the tablets may be coated with a material such as glyceryl
monostearate or glyceryl distearate, to delay absorption in the
gastrointestinal tract.
[0039] Capsules for oral use include hard capsules on which the
active ingredient is mixed with a solid diluent, and soft capsules
wherein the active ingredient is mixed with water or an oil such as
peanut oil, liquid paraffin or olive oil.
[0040] For intramuscular, intraperitoneal, subcutaneous and
intravenous use, the compounds of the invention will generally be
provided in sterile aqueous solutions or suspensions, buffered to
an appropriate pH and isotonicity. Suitable aqueous vehicles
include Ringer's solution and isotonic sodium chloride. Aqueous
suspensions according to the invention may include suspending
agents such as cellulose derivatives, sodium alginate,
polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such
as lecithin. Suitable preservatives for aqueous suspensions include
ethyl and n-propyl p-hydroxybenzoate.
[0041] The vaccines prepared by the process of the present
invention may also be presented as liposome formulations.
[0042] The present invention is now described with reference to the
following examples and to the figures in which:-
[0043] FIG. 1 shows the kinetics of growth of H. pylori CCUG 17874
in BB containing Tryptone and peptamin.
[0044] FIG. 2 shows the kinetics of growth of H. pylori CCUG 17874
in simplified BB containing Soytone.
[0045] FIG. 3 shows the kinetics of growth H. pylori CCUG 17874 in
simplified BB containing Hysoy.
[0046] FIG. 4 shows a VacA immunoblot wherein lane 1 is molecular
weight marker, lane 2 VacA standard 750 ng, lane 3 VacA standard
500 ng, lane 4 VacA standard 400 ng, lane 5 VacA standard 150 ng,
lane 6 VacA standard 20 ng, lane 7 VacA produced at the end of
fermentation, lane 8 VacA produced after 48 hours of culture, lane
9 VacA produced after 30 hours of culture, lane 10 VacA produced
after 23 hours of culture.
[0047] FIG. 5 shows a VacA immunoblot wherein lane 1 is molecular
weight marker, lane 2 VacA standard 750 ng, lane 3 VacA standard
500 ng, lane 4 VacA standard 400 ng, lane 5 VacA standard 150 ng,
lane 6 VacA standard 20 ng, lane 7 VacA produced at the end of
fermentation, lane 8 VacA produced after 31.5 hours of culture,
lane 9 VacA produced after 30 hours of culture, lane 10 VacA
produced after 24 hours of culture.
[0048] FIG. 6 shows a VacA immunoblot wherein lane 1 is molecular
weight marker, lane 2 VacA standard 750 ng, lane 3 VacA standard
500 ng, lane 4 VacA standard 400 ng, lane 5 VacA standard 20 ng,
lane 6 VacA produced at the end of fermentation, lane 7 VacA
produced after 48 hours of culture, lane 8 VacA produced after 31
hours of culture, lane 9 VacA produced after 24.5 hours of culture,
lane 10 VacA standard 150 ng.
[0049] FIG. 7 shows the kinetics of growth of H. influenzae b in
simplified Franz medium containing Soytone or Proteose peptone.
[0050] FIG. 8 shows the kinetics of growth of N. meningitidis C in
Watson medium containing Casaminoacids or Amisoy.
EXAMPLES
[0051] All references are herein incorporated by reference.
Example I
[0052] Helicobacter pylori is a curved gram-negative microaerobic
bacterium isolated about 10 years ago and is associated with type B
gastritis in humans. This bacterium colonizes the human gastric
mucosa and establishes a chronic infection that may result in
gastric and duodenal ulcers (Blaser, M. J., (1990), J.Infect.Dis.,
161, 629-633) and can be a risk factor for the development of
gastric carcinoma (Parsonnet, J. et al., (1991), New Engl. J. Med.,
325,1127-1131).
[0053] In the long term, the infection and the diseases could be
prevented and treated by vaccination. Currently, several factors
involved in bacterial adhesion, colonisation and virulence have
been identified. One of the most interesting factors involved in
the disease is the vacuolating cytotoxin (VacA) that causes massive
vacuolization in several mammalian cell lines (Leunk, R. D.,
(1991), Rev. Infect. Dis., 13(suppl.8), S683-S689). Vacuoles have
also been observed in the gastric epithelia of patients with
chronic gastritis (Tricottet, V. et al., (1986), Ultrastruct.
Pathol., 10, 113-117). This protein has been shown to cause
ulceration in mice (Telford, J. L. et al., (1994), J. Exp. Med.,
179, 1653-1658) and is a vaccine candidate. The purified cytotoxin
is a protein of 87-94 kD that can be purified in very small amounts
from bacterial culture supernatant.
[0054] Material and Methods
[0055] Bacterial Strain.
[0056] The Helicobacter pylori CCUG 17874 (type strain, Culture
Collection, University of Goteborg) was used.
[0057] Media and Supplements.
[0058] Brucella Broth (tryptone 10 g 1.sup.-1, peptamin 10 g
1.sup.-1, dextrose 1 g 1.sup.-1, yeast extract 2 g 1.sup.-1, sodium
chloride 5 g 1.sup.-1 and sodium bisulfite 0.1 g 1.sup.-1)
(BB)(Difco) supplemented with 2 g 1.sup.-1 of (2,6
di-0-methyl)-b-cyclodextrin (CD) (Teijin Lim. Tokyo, Japan) and 20
mg/L of streptomycin was used as liquid medium for comparison
purpose. Hysoy or Soytone were used at a concentration of 10 g/L
instead of tryptone and peptamin present in BB.
[0059] Preservation.
[0060] Frozen aliquots for inocula were prepared from flask
cultures of 2.times.10.sup.8 CFU ml1.sup.-1 diluted 1:2 with a
solution composed of glycerol 40%, fetal calf serum (FCS) (Hyclone,
Logan, Utah) 20% and 0.4% CD. The suspension obtained was
distributed in 3 ml vials and stored at -80.degree. C. and used as
starting frozen vials for comparison with new frozen vials prepared
substituting FCS with Soytone 20%.
[0061] Growth in Liquid Medium.
[0062] Initial cultures were performed in 500 ml Erlenmeyer flasks
containing 100 ml of liquid medium. Cultures were inoculated with 3
ml of frozen stocks and incubated at 36.degree. C. for 36 hours
with shaking (100 rpm, 2.5 cm throw) in a microaerobic environment.
Flasks were placed inside an anaerobic jar where BBL Campy Pak
envelopes (Becton Dickinson) were used to generate the proper
conditions. These cultures were then used to inoculate 1000 ml
flasks containing 250 ml of medium and incubated in the same
conditions mentioned above and used to inoculate the
bioreactors.
[0063] Culture Vessels and Growth Conditions.
[0064] Batch fermentations were carried out in 7 litres bioreactors
(MBR Bioreactors AG, 8620 Wetzikon, CH) containing 5 I of medium.
All cultures were grown at 36.degree. C. The pH values were not
controlled. The dissolved oxygen tension (DOT) was maintained
automatically at the pre-set level (3%) by a two step procedure.
First, air flow rate was increased from 0.1 up to 0.5 l 1.sup.-1
min.sup.-1 to satisfy the increasing O.sub.2 demand of the culture.
If further increases were necessary, they were obtained by
supplying pure O.sub.2 up to a maximum of 0.4 l 1.sup.-1
min.sup.-1. During the first 12 hours of growth, a constant flow of
N.sub.2 and C.sub.2 was maintained equal to 0.2 l 1.sup.-1
min.sup.-1 and 0.02 l 1.sup.-1 min .sup.-1 respectively. The
agitation speed was maintained at 130 rpm. The agitator shaft was
equipped with two Rhuston turbines having a diameter of 7 cm and
the diameter of the bioreactor was 17 cm.
[0065] Glucose Feed.
[0066] A 50% glucose solution was added at time 0 to give a final
concentration of 5 g/L. Another addition of 5 g/L was made when the
OD was in a range 2-3.
[0067] Biomass Determination.
[0068] Growth was monitored by optical density at 590 nm against a
water blank (Perkin Elmer 35 spectrophotometer), light path of 1
cm. Purity checks of the samples were made by Gram staining.
[0069] Analysis of the VacA Protein.
[0070] At determined time points during the fermentation, culture
samples were centrifuged (Biofuge A, Heareus) at 8,300.times.g per
10 min. The supernatants were precipitated with trichloroacetic
acid and subjected to 9% SDS-Page using a BioRad Mini Protean II
apparatus. Proteins were transferred to nitrocellulose filters
(Schleicher & Schuell) and then incubated overnight with
polyclonal antisera raised against the VacA protein (Telford, J. L.
et al., (1994), J. Exp. Med., 179, 1653-1658). After incubation for
2 hours with a horseradish-peroxidase conjugated secondary antibody
(Sigma), the immunoreactive bands were visualized by
4-chloro-naphtol staining.
[0071] Results
[0072] Brucella Broth is a complex medium composed of tryptone 10 g
1.sup.-1, peptamin 10 g 1.sup.-1, dextrose 1 g 1.sup.-1, yeast
extract 2 g 1.sup.-1, sodium chloride 5 g 1.sup.-1 and sodium
bisulfite 0.1 g 1.sup.-1. This medium has been described in many
articles as capable of supporting the growth of H. pylori only when
supplemented with blood derivatives (Cover, T. L. and Blaser, M. J.
(1992), J. Biol. Chem., 267, 10570-10575; Shahamat, M. et al.,
(1991), J. Clin. Microbiol., 29, 2835-2837; Buck, G. E. and Smith,
J. S. (1987), J. Clin. Microbiol., 25, 597-599; and Morgan, D. R.
et al., (1987), J. Clin. Microbiol., 25, 2123-2125). A substantial
simplification of H. pylori growth media was obtained recently when
it was discovered that cyclodextrins could be used in the place of
blood derivatives (Olivieri, R. et al., (1993), J. Clin.
Microbiol., 31, 160-162). The growth of H. pylori using this
simplified medium and glucose feed is reported in FIG. 1. Glucose
feeds were used to ensure that the carbon and energy source was
always present in the medium.
[0073] When H. pylori was cultured in these conditions, the
production of VacA in the medium was measured as described above.
The results are shown in FIG. 5 and FIG. 6.
[0074] The use of Soytone and Hysoy in the growth media gave the
results reported in FIGS. 2 and 3 respectively. VacA production is
reported in FIG. 5 when Soytone was used and in FIG. 6 when Hysoy
was used.
[0075] The results show improvements to the growth media and
fermentation conditions of Helicobacter pylori growth and in the
production of the vacuolating cytotoxin (VacA).
Example II
[0076] Haemophilus influenzae are small, non motile, gram negative
bacteria that are the major cause of bacterial meningitidis in
children. These microorganisms are primarily invasive rather than
toxigenic. They are inhabitants of the respiratory tract (commensal
as well pathogenic) and they have antiphagocytic polysaccharides
capsules.
[0077] Materials and Methods
[0078] Bacterial Strain
[0079] Haemophilus Influenzae B ATCC 10211
[0080] Media and Supplements
[0081] The preparation of the media, involves use of different
solutions as follows:
[0082] "Franz A Medium" Preparation
1 Component Amount per litre Purified water 800 mL Glutamic acid
1.6 g +/- 0.01 g Na2HPO.sub.4 12H2O 5.03 g +/- 0.05 g KCl 0.892 g
+/- 0.008 g NaCl 6.005 g +/- 0.06 g NH4Cl 1.25 g +/- 0.01 g
Purified water QS to 1.0 litre 3N NaOH as required for pH =
8.2.
[0083] The above components are dissolved with mixing. 3N NaOH is
used to pH the solution to pH=8.2
[0084] Ultrafiltered Soytone Preparation
2 Component Amount per litre Soytone 33.3 g +/- 0.03 g Purified
water QS to 1.0 litre
[0085] This solution was ultrafiltered through a 30 kD TFF
apparatus.
[0086] Ultrafiltered Proteose Peptone
3 Component Amount per litre Proteose peptone 33.3 g +/- 0.03 g
Purified water QS to 1.0 litre
[0087] This solution was ultrafiltered through a 30 kD TFF
apparatus
[0088] Ultrafiltered YE Preparation
4 Component Amount per litre yeast extract 100 g +/- 0.02 g
Purified water QS to 1.0 litre
[0089] The yeast extract used was HY YEST available from Quest. The
solution was ultrafiltered through a 10 kD TFF apparatus.
[0090] 50% Glucose Solution
5 Component Amount per litre glucose (anhydrous) 500 g +/- 5 g
Purified water QS to 1.0 litre
[0091] NAD 0.1% Solution
6 Component Amount per litre NAD 1.0 g +/- 0.005 g TRIS 1.21 g +/-
0.01 g Purified water QS to 1.0 litre HCl 37% as required to pH 7.4
.+-. 0.2
[0092] Hemin 0.4% Solution
7 Component Amount per litre Hemin 4 g +/- 0.02 g 0.2N NaOH QS to
1.0 litre
[0093] The hemin used is preferably chemically synthesised.
Chemically synthesised hemin is commercially available from
Fluka
[0094] 550 ml of Franz A was mixed with 450 ml of ultrafiltrated
soytone or Proteose Peptone to obtain one litre of Hib basal medium
which was sterilized by autoclaving at 121.degree. C. for 30
minutes.
[0095] After cooling, 10 ml/L glucose solution, 20 ml/L of YE, 2
ml/L NAD solution, sterilized by filtration, were added (with the
additions mentioned before) and the medium called "Hib Complete
medium".
[0096] Growth in Liquid Media
[0097] Pre-warmed, unbaffeled shake flasks (500/150) were
inoculated with 1.0 mL of a working stock vial each. The shake
flasks were placed in an (1 inch throw) incubator-shaker at
35+/-1.degree. C. at 150 RPM for 6 hours.
[0098] After 6 hours, the appropriate amount of the shake flask was
transferred into one 2 L litre unbaffeled shake flask containing
0.5 L of pre-warmed "Hib complete medium". The shake flask was
placed in an (1 inch throw) incubator-shaker at 35+/-1.degree. C.
at 200 rpm for 9 hours then the content was transferred into a
sterile inoculation and the inoculate then transferred to the
fermenter.
[0099] Culture Vessels and Growth Conditions
[0100] Batch fermentations were carried out in 30 litres
bioreactors (MBR Bioreactors AG, 8620 Wetzikon, CH) containing 20 l
of medium.
[0101] The cultures were growing at 35.degree. C. and 2 psi back
pressure. The pH was controlled to 7.3 with 3 N NaOH. The initial
agitation rate was set at minimum of 150 rpm, and bottom aeration
at 10 L/minute. DOT was maintained at 35% by rpm control in a range
150-400, then supplementing with oxygen if necessary. Antifoam was
added manually to control foaming. Residual concentration of
glucose was detected and when it was around 2 g/L, 0.2 litres of
glucose solution were added.
[0102] Biomass Determination.
[0103] Growth was monitored by optical density at 590 nm against a
water blank (Perkin Elmer 35 spectrophotometer), light path of 1
cm. Purity checks of the samples were made by Gram staining.
[0104] Analysis of Hib PS.
[0105] This analysis was performed by Rocket immuno electrophoresis
as described by Weeke, B., Scand. J. immunol. 2, 37-46, (1973),
herein incorporated by reference.
[0106] Results
[0107] The growth curves obtained with Soytone and with Proteose
peptone are compared in FIG. 7. The yield of Hib PS was 600 mg/L
and 150 mg/L in the media containing Soytone and Proteose peptone
respectively. Although the growth curves are quite similar using
the two media, there is a fourfold increase in the yield of Hib PS
using Soytone. The use of the culture media containing the
vegetable derived proteinaceous material leads to an increased
yield of polysaccharides, such as Hib PS, compared to the use of a
culture medium containing animal derived proteinaceous
material.
Example III
[0108] C. diphtheriae are gram positive, rod like microorganisms,
which arrange themselves in palisades. C. diphtheriae
lysogenization by a bacteriophage causes the synthesis of a potent
toxin whose expression is regulated by iron concentration.
[0109] Materials and Methods
[0110] Bacterial Strain
[0111] C. diphtheriae CN 2000
[0112] Media and Supplements
[0113] The preparation of the media, involves use of different
solutions as follows:
[0114] A. Yeast Extract (YE) and Casamino Acids (CAA)
Ultrafiltered
8 Component Amount g/L Purified water 800 ml Yeast Extract 20 g/L
Casamino Acids 10 g/L Purified water QS to 1 L
[0115] This solution was ultrafiltered through a 10 kD TFF
apparatus and the penneate added to the deferration vessel.
[0116] A.A Yeast Extract (YE) and Soytone Ultrafiltered
9 Component Amount g/L Purified water 800 ml Yeast Extract 20 g/L
Soytone 10 g/L Purified water QS to 1 L
[0117] This solution was ultrafiltered through a 10 kD TFF
apparatus and the permeate added to the deferration vessel.
[0118] A.B Yeast Extract Ultrafiltered
10 Component Amount g/L Purified water 800 ml Yeast Extract 30 g/l
Purified water QS to 1 L
[0119] This solution was ultrafiltered through a 10 kD TFF
apparatus and the permeate added to the deferration vessel.
[0120] A.1 Deferration
[0121] The following components were introduced into an agitated
vessel
11 Component Amount UF (YE + CAA) solution 1 L KH.sub.2PO.sub.4 5
g/L CaCl.sub.2-2H.sub.2O, 50% (w/v) 2 mL/L L-tryptophan, 1% (w/v) 5
mL/L (0.05 g/L) 3N NaOH (enough to correct pH to 7.4)
[0122] UF indicates that the solution is ultrafiltered.
[0123] With agitation, heat the solution to 100.degree. C. Hold at
100.degree. C. for 1 minute, then cool medium to 37.degree. C.
Filtration can commence once below 37.degree. C. After filtration,
the medium is described as "CY base medium with CAA".
[0124] A.1.A Deferration
[0125] The following components were introduced into an agitated
vessel
12 Component Amount UF (YE + Soytone) solution 1 L KH.sub.2PO.sub.4
5 g/L CaCl.sub.2-2H.sub.2O, 50% (w/v) 2 mL/L L-tryptophan, 1% (w/v)
5 mL/L (0.05 g/L) 3N NaOH (enough to correct pH to 7.4)
[0126] With agitation, heat the solution to 100.degree. C. Hold at
100.degree. C. for 1 minute, then cool medium to 37.degree. C.
Filtration can commence once below 37.degree. C. After filtration,
the medium is described as "CY base medium with Soytone".
[0127] A.1.B Deferration
[0128] The following components were introduced into an agitated
vessel
13 Component Amount UF YE solution 1 L KH.sub.2PO.sub.4 5 g/L
CaCl.sub.2-2H.sub.2O, 50% (w/v) 2 mL/L L-tryptophan, 1% (w/v) 5
mL/L (0.05 g/L) 3N NaOH (enough to correct pH to 7.4)
[0129] With agitation, heat the solution to 100.degree. C. Hold at
100.degree. C. for 1 minute, then cool medium to 37.degree. C.
Filtration can commence once below 37.degree. C. After filtration,
the medium is described as "CY base medium with YE only".
[0130] B. "Supplements" Solution
[0131] B1) Solution A
14 Component Amount per litre MgSO.sub.4-7H.sub.2O 225 g
Beta-alanine 1.15 g Nicotinic acid 1.15 g Pimelic acid 0.075 g
CuSO.sub.4 0.50 g ZnSO.sub.4-7H.sub.2O 0.40 g MnCl.sub.2-4H.sub.2O
0.15 g HCl, 37% 30 mL Purified water QS to 1.0 litres
[0132] B2) Solution B
15 Component Amount per litre Purified water 800 mL L-cystine 200 g
HCl, 37% 200 mL
[0133] The solutions are mixed individually for 10 minutes. After
dissolution, 20 mL of Solution A and 10 mL of Solution B are mixed
and filtered through a 0.2 micron filter. The solution is stored at
4.degree. C. covered from light.
[0134] Flasks Sterilization
[0135] Once deferrated, filtered media (A.1 or A.1.A or A.1.B) are
loaded into the flasks. Sterilize the flasks for 25 minutes at
121.degree. C.
[0136] Post-Sterilization Adjustments
16 1. Add "50% Maltose" 30 ml/L 2. Add "Supplements" 3.0 ml/L
[0137] Media with 1+2 above: "Complete CY medium with CAA or with
Soytone or with YE only"
[0138] Growth in Liquid Media
[0139] 500 ml unbaffeled shake flasks each with 100 ml of medium,
were inoculated with 1.0 mL of a working stock vial.
[0140] The shake flasks were placed in an (1 inch throw)
incubator-shaker at 35+/-1.degree. C. at 100 rpm for 5 hours. After
5 hours, the agitation is increased to 250 rpm for 43 hours.
[0141] The process has two distinct phases: 1) Exponential growth),
and 2) Production phase. The transition from the two phases is
gradual and is marked by reductions in cell growth rate and oxygen
demand.
[0142] Biomass Determination.
[0143] Growth was monitored by optical density at 590 nm against a
water blank (Perkin Elmer 35 spectrophotometer), light path of 1
cm. Purity checks of the samples were made by Gram staining.
[0144] Results
[0145] After 48 hours of incubation, the OD and the Lf/ml in the
flasks were:
17 medium with CAA. OD = 7.5 Lf/ml = 50 medium with Soytone. OD =
7.87 Lf/ml = 60 medium with YE only OD = 8.07 Lf/ml = 60
Example IV
[0146] Neisseria meningitidis are non motile gram negative cocci,
most often growing in pairs but occasionally in tetrads or
clusters. They have antiphagocytic polysaccharides capsules which
is the basis for serogroups.
[0147] Materials and Methods
[0148] Bacteria Strain
[0149] Neisseria meningitidis. C11
[0150] Media and Supplements
[0151] Preparation of the media involves use of different solutions
as follows:
[0152] "Franz medium" Preparation
[0153] Inoculum Shake Flask Medium Preparation
18 Component Amount per litre Purified water 800 mL Glutamic acid
1.6 g Na2HPO.sub.42H2O 15.5 g KCl 0.09 g NH4Cl 1.25 g Purified
water QS to 1.0 litre 3N NaOH as required for pH = 7.6
[0154] Dissolve the above components with mixing. Sterilize by
autoclaving at 121.degree. C., 30 minutes. After cooling, the "50%
Glucose" and "Men C Supplements" are added as below.
19 "50% Glucose" 10 mL/L +/- 0.3 mL "Men C Fermentation
Supplements" 20 mL/L +/- 0.3 mL
[0155] With the addition of the glucose and supplements solutions,
the medium can be called "Franz Complete medium".
[0156] Preparation of the 20 L Fermenter
[0157] A. The following components must be introduced to a mixing
vessel and dissolved to create 20 L of Watson base medium with
CAA:
20 Component Amount per litre Purified water 800 mL Glutamic acid 1
g +/- 0.01 g Na.sub.2HPO.sub.42H.sub.2O 3.25 g +/- 0.03 g KCl 0.09
g +/- 0.001 g Casamino acid 10 g +/- 0.1 g Purified water QS to 1.0
litre 3N NaOH as required for pH = 7.6
[0158] A.A The following components must be introduced to a mixing
vessel and dissolved to create 20 L of Watson base medium with
Amisoy:
21 Component Amount per litre Purified water 800 mL Glutamic acid 1
g +/- 0.01 g Na.sub.2HPO.sub.42H.sub.2O 3.25 g +/- 0.03 g KCl 0.09
g +/- 0.001 g Amisoy 10 g +/- 0.1 g Purified water QS to 1.0 litre
3N NaOH as required for pH = 7.6
[0159] B. Fermenter Sterilization
[0160] Sterilize the fermenter for 25 minutes at 121.degree. C.
After sterilization, set the following conditions: temperature,
35.degree. C.; aeration at 10 L/minute; agitation at 150 rpm, and
backpressure at 0.2 bar. After cooling the fermenter to 35.degree.
C., and take a sample to measure pH, adjust the fermenter pH probe
calibration and then adjust the medium pH 25 to 7.6.
[0161] C. Post-Sterilization Adjustments
22 1. Add "50% Glucose" 10 ml/L 2. Add "Men C Supplement" 20
ml/L
[0162] Medium with 1+2 above: "Watson Complete medium"
[0163] A. "50% Glucose" Solution
23 Component Amount per litre glucose (anhydrous) 500 g +/- 5 g
Purified water QS to 1.0 L
[0164] This solution is sterilized by autoclaving at 121.degree. C.
for 30 minutes.
[0165] B. "Men C Supplements" Solution
[0166] B.1 Ultrafiltered YE
24 Component Amount Purified water QS to 1 L Yeast Extract 125
g/L
[0167] This solution is ultrafiltered through a 10 kD TFF
apparatus. The retentate is discarded after the requisite permeate
has been collected, and the permeate is added to the vessel
containing the supplement.
[0168] B.2
25 Component Amount per litre of UF YE MgSO.sub.4-7 H.sub.2O 30 g
+/- 0.5g L-cystein-HCl 1.5 g +/- 0.2 g
[0169] Add the chemicals to 1 L UF YE, mix for 10 minutes, filter
sterilized through a 0.2 micron filter and transfer to the
fermenter.
[0170] C. 3 N NaOH
[0171] 3 N NaOH=12% (w/v) NaOH=120 g/L NaOH
[0172] D. Antifoam
[0173] The antifoam used is Dow Corning 1510. It is sterilized by
autoclaving at 121.degree. C. for 30 minutes
[0174] Growth in Liquid Media
[0175] A 500 millilitre flask is inoculated with 1.0 mL of a
working stock vial. The shake flask contains 150 mL of complete
"Franz medium".
[0176] The inoculated shake flasks is placed in an (1 inch throw)
incubator-shaker at 35+/-1.degree. C. at 150 rpm. After 10 hours,
the flask is aseptically sampled. The optical density should be
between 1.3-3.3 (at 590 nm) if so, the four 2 L shake flasks are
each inoculated with the appropriate volume from the 150 mL shake
flask.
[0177] Each 2 L shake flask contains 0.2 L of pre-warmed "Franz
Complete medium".
[0178] The shake flasks are placed in an (1 inch throw)
incubator-shaker at 35+/-1.degree. C. at 200 rpm. After 10 hours,
the contents of each shake flask are transferred into the sterile
inoculation can and inoculate the fermenter.
[0179] Culture Vessel and Growth Conditions
[0180] Batch fermentations were carried out in 30 litres
bioreactors (MBR Bioreactors AG, 8620 Wetzikon, CH) containing 20 L
of medium.
[0181] The cultures were growing at 35.degree. C. and
14.times.10.sup.3 N/m.sup.2 (2 psi) back pressure. The pH was
controlled to 7.3 with 3 N NaOH. The initial agitation rate was set
at minimum of 150 rpm, and bottom aeration at 10 L/minute. DOT was
maintained at 35% by rpm control in a range 150-400, then
supplementing with oxygen if necessary. Antifoam was added manually
to control foaming. Residual concentration of glucose was detected
and when it was around 2 g/L, 0.2 litres of glucose solution were
added.
[0182] Biomass Determination.
[0183] Growth was monitored by optical density at 590 nm against a
water blank (Perkil Elmer 35 spectrophotometer), light path of 1
cm. Purity checks of the samples were made by Gram staining.
[0184] Analysis of MenC PS
[0185] Quantitative estimation of the polysaccharides was performed
analyzing the sialic acid content according to the method reported
in Biochimica and Biophysica Acta (1957), 21, 610, by Lars
Svennerholm.
[0186] Results
[0187] The growth curves obtained with Amisoy and with Casamino
acids are compared in FIG. 8. The yield in MenC PS was 307 mg/L and
345 mg/L in the media containing Amisoy and Casamino acids
respectively. The growth curves and PS production are quite similar
using the two media.
[0188] It will be understood that the invention is described above
by way of example only and modifications may be made within the
scope of the invention as defined in the appended claims.
Example V
[0189] Clostridium tetani is a slender bacillus measuring 2 .mu.m
in length and 0.3-0.5 .mu.m in width. It often exists in the form
of a rather long filament-like cell. When spores are formed, the
bacillus assumes the characteristic drumstick appearance. It is a
mobile organism, gram positive, but its gram stain can become
variable or even negative in aging cultures. Clostridium tetani is
a strict anaerobe and produces two exotoxins. One of these, the
tetano spasmin, is a neurotoxin responsible for the whole clinical
picture of the disease.
[0190] Materials and Methods
[0191] Bacterial Strain
[0192] Clostridium tetani Harvary Y-VI-3.
[0193] Media
[0194] The seed cultures were prepared using the medium reported
below expressed in g/L:
26 Component Amount g/L N-Z Soy 15.0 Glucose 5.5 Yeast extract 5.0
NaCl 2.5 L-Cysteine 0.5 Sodium thioglycollate 0.5 Agar 0.75 PH =
7.1
[0195] The production media were prepared as modification of the
Mueller-Miller medium described in the WHONSQ/GEN/94 (1990). In
this medium, beef heart infusion and casein solution are used, in
the modified media reported below, expressed in g/L, Hysoy and
Soytone were used instead of beef heart infusion and casein
solution:
27 Component Amount g/L Glucose-H.sub.2O 12.1 NaCl 2.5
Na.sub.2HPO.sub.4-12H.sub.- 2O 2.5 KH.sub.2PO.sub.4 0.15
MgSO.sub.4-7H.sub.2O 0.15 Amino acids solution 17.5 ml Vitamine
solution 4.2 ml NaOH 5M 4.0 ml FeSO.sub.4-7 H.sub.2O (1% sol.) 4.0
ml Soy derivatives 20.0 PH = 7.3
[0196] Sterilised by autoclaving at 120.degree. C. for 20 min
28 Component Amino acids solution: L-Tyrosine 28.51 g/L Uracil-
0.142 g/L L-Cystein- 14.25 g/L HCl 37% 131.6 ml/L Vitamin solution:
Ca pantithenate 238.1 mg/L Thiamine 59.7 mg/L Pyridoxin 59.7 mg/L
Riboflavin 59.7 mg/L Biotin 0.73 mg/L Ethanol 256.4 ml/L
[0197] Growth in Liquid Media
[0198] Two 25 ml tubes, containing 15 ml of seed medium, were
inoculated with 0.5 ml each of working seed vial and incubated at
35.degree. C. for 29 hrs in anaerobic jar where a Gas generating
kit (OXOID) was used. A second series of tubes were inoculated by
1.5 ml of the first tubes and incubated in the same conditions
reported above for 24 hrs.
[0199] 7 ml of these cultures were used to inoculate 100 ml tubes
containing 75 ml of the same medium. These tubes were incubated in
the same conditions reported above for 24 hrs.
[0200] The entire content of these tubes were used to inoculate
5000 ml beakers containing 2500 ml of the production medium.
[0201] Biomass Determination
[0202] Growth was monitored by optical density at 590 nm against a
water blank (Pharmacia spectrophotometer), light path of 1 cm.
Purity checks of the samples were made by Gram staining.
[0203] Results
[0204] After 186 hrs of incubation, the OD and the Lf/ml in the
breakers were:
29 Medium with Hysoy OD = 1.08 Lf/ml = 60 Medium with Soytone OD =
0.74 Lf/ml = 60 Medium with beef heart infusion and OD = 1.236
Lf/ml = 60 casein solution
Example VI
[0205] Bordetella pertussis is a gram negative coccobacillus about
0.5 .mu.m in diameter and 0.5 to 2 .mu.m in length. Its nutritional
requirements are simple, and it does not utilize sugars. It is
extremely sensitive to fatty acids and survives poorly without
protective factors.
[0206] Materials and Methods
[0207] Bacterial Strain
[0208] Bordetella pertussis 9K/129G (Pizza, M., et al. (1989)
Science, 246, 497-500).
[0209] Media
[0210] The seed and production cultures were prepared using CL
medium (Imaizumi, A., et al. (1983) Infection and Immunity, 41 (3),
1138-1143) reported below expressed in g/L:
30 Component Amount g/L Sodium L-glutamate 10.7 L-proline 0.24 NaCl
2.5 KH.sub.2PO.sub.4 0.5 KCl 0.2 MgCl.sub.2-6H.sub.2O 0.1
CaCl.sub.2 0.02 Tris 6.1 L-Cysteine* 0.04 FeSO.sub.4-7 H.sub.2O*
0.01 Niacin* 0.004 Glutathione reduced* 0.15 Ascorbic acid* 0.4
Casaminoacid 10.0 Dimethyl-B-cyclodextrin 1.0 PH adjusted to 7.6
with HCl. *sterilized by filtration and then added aseptically to
the autoclaved medium
[0211] The modified medium contained 10 g/l of N-Z soy instead of
Casaminoacid.
[0212] Growth in Liquid Media
[0213] 500 ml unbaffeled shake flasks each with 100 ml of CL medium
or the modified one, were inoculated with 3.0 mL of a working stock
vial.
[0214] The shake flasks were placed in an (1" throw)
incubator-shaker at 35+/-1.degree. C. at 100 RPM for 12 hours.
After 12 hours, the agitation was increased to 250 RPM for another
16 hours.
[0215] Biomass Determination
[0216] Growth was monitored by optical density at 590 mm against a
water blank (Pharmacia spectrophotometer), light path of 1 cm.
Purity checks of the samples were made by Gram staining.
[0217] Analysis of PT
[0218] This analysis was performed by ELISA (Nencioni, L., et al.
(1990) Infect. Immun., 58, 1306-1315).
[0219] Results
[0220] After 28 hrs of incubation, the OD and PT (mg/L) in the
flasks were:
31 medium with CAA. OD = 2.31 PT = 2.4 mg/L Medium with NZ-soy. OD
= 1.99 PT = 2.25 mg/L
* * * * *