U.S. patent application number 15/738889 was filed with the patent office on 2018-07-05 for lactobacillus paracasei for the production of conjugated linoleic acid, nutritional and pharmaceutical preparations containing it and uses thereof.
This patent application is currently assigned to ALFASIGMA S.P.A.. The applicant listed for this patent is ALFASIGMA S.P.A.. Invention is credited to Marina ELLI, Pierluigi MANGINO, Annalisa SFORZINI, Giuseppe Claudio VISCOMI.
Application Number | 20180187274 15/738889 |
Document ID | / |
Family ID | 54347708 |
Filed Date | 2018-07-05 |
United States Patent
Application |
20180187274 |
Kind Code |
A1 |
VISCOMI; Giuseppe Claudio ;
et al. |
July 5, 2018 |
LACTOBACILLUS PARACASEI FOR THE PRODUCTION OF CONJUGATED LINOLEIC
ACID, NUTRITIONAL AND PHARMACEUTICAL PREPARATIONS CONTAINING IT AND
USES THEREOF
Abstract
The present invention refers to a new strain belonging to the
species Lactobacillus paracasei able to convert linoleic acid into
conjugated linoleic acid. The present invention refers also to
nutritional or food preparations and/or pharmaceutical
compositions, comprising the strain Lactobacillus paracasei, useful
in the treatment and/or prevention of pathologies and/or
physiological states related to conjugated linoleic acid deficiency
or in the cases wherein the use of a probiotic is beneficial.
Inventors: |
VISCOMI; Giuseppe Claudio;
(Bologna (BO), IT) ; SFORZINI; Annalisa; (Bologna,
IT) ; MANGINO; Pierluigi; (Bologna, IT) ;
ELLI; Marina; (Piacenza PC, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALFASIGMA S.P.A. |
Milano |
|
IT |
|
|
Assignee: |
ALFASIGMA S.P.A.
Milano
IT
|
Family ID: |
54347708 |
Appl. No.: |
15/738889 |
Filed: |
June 30, 2016 |
PCT Filed: |
June 30, 2016 |
PCT NO: |
PCT/EP2016/065245 |
371 Date: |
December 21, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 33/135 20160801;
A61K 35/747 20130101; C12R 1/225 20130101; A61P 3/00 20180101; C12N
1/20 20130101; A61K 9/2095 20130101; A61K 9/009 20130101; A23V
2002/00 20130101 |
International
Class: |
C12R 1/225 20060101
C12R001/225; A61K 35/747 20060101 A61K035/747; A61K 9/00 20060101
A61K009/00; A61K 9/20 20060101 A61K009/20; A23L 33/135 20060101
A23L033/135 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 7, 2015 |
IT |
UB 2015 A 002376 |
Claims
1. A bacterial strain belonging to the species Lactobacillus
paracasei filed at the Coordinated Collection of
Microorganism-BCCM/LMG Bacteria Collection, Microbiology
Laboratory-Ghent University, registered with the number LMG
S-26420, characterized in that it converts linoleic acid in
conjugated linoleic acid in a percentage higher than 30%.
2. A bacterial strain Lactobacillus paracasei LMG S-26420 according
to claim 1, characterized in that it converts linoleic acid into
the isomers with biological activity of conjugated linoleic acid
c9-t11 e t9-t11, wherein their sum is in a percentage higher than
30% if compared to the other isomers.
3. A process for the production of the strain Lactobacillus
paracasei LMG S-26420 of claim 1 characterized by the following
steps: inoculating the strain Lactobacillus paracasei LMG S-26420
in a volumetric percentage from 0.1% to 10%, in a suitable culture
medium at a temperature from 30.degree. C. to 37.degree. C. at pH
values between 4.5 and 7.5 for a time from 6 to 15 hours;
separating the bacterial mass from the culture broth by
centrifugation; storing the bacterial mass at a temperature lower
than 0.degree. C., or lyophilising.
4. A process for the production of the bacterial strain
Lactobacillus paracasei LMG S-26420 according to claim 3,
comprising the step of lyophilizing in the presence of
cryoprotectants selected among soluble carbohydrates.
5. A process according to claim 4 for the production of the
lyophilic bacterial strain Lactobacillus paracasei LMG S-26420 in
the presence of trealose.
6. A process according to claim 3, wherein the strain Lactobacillus
paracasei LMG S-26420 is obtained with a number of colony forming
units higher than 109 for millilitre of culture, and the biomass in
an amount higher than 4 grams per litre of culture.
7. A nutritional or food or pharmaceutical composition comprising a
cell amount of the bacterial strain Lactobacillus paracasei LMG
S-26420 from 1.times.108 to 5.times.1011, together with acceptable
excipients in form of tablets, granules for sachets, capsules or
liquid suspensions.
8. A nutritional or food or pharmaceutical composition according to
claim 7 comprising prebiotics, vitamins, mineral salts.
9. A nutritional or food or pharmaceutical composition according to
claim 8, wherein the prebiotics are selected from the group
consisting of fructo-oligosaccharides, inulines,
galacto-oligosaccharides, xylo-oligosaccharides, and
isomalto-oligosaccharides and the vitamins are selected from the
group consisting of vitamins E and B.
10. A nutritional or food or pharmaceutical composition according
to claim 7 comprising bifidobacteria.
11. Method of treating or preventing pathologies or diseases
related to a deficiency of conjugated linoleic acid and in all
cases where a probiotic is beneficial in a subject in need thereof,
said method comprising administering to said subject a
pharmaceutical effective amount of a nutritional or food or
pharmaceutical composition comprising the bacterial strain
Lactobacillus paracasei LMG S-26420.
12. Method of treating and/or preventing inflammatory diseases,
intestinal inflammatory pathologies, oxidative stress, tumour,
autoimmune diseases, diabetes and atherosclerosis in a subject in
need thereof with a nutritional or food or pharmaceutical
composition comprising the bacterial strain Lactobacillus paracasei
LMG S-26420, said method comprising: administering to said subject
a pharmaceutical effective amount of said nutritional or food or
pharmaceutical composition comprising the bacterial strain
Lactobacillus paracasei LMG S-26420.
13. Method of treating diseases related to bacterial dysbiosis in a
subject in need thereof with the nutritional or food or
pharmaceutical composition according to claim 7 comprising
lactobacilli belonging to different strains and/or Bifidobacteria,
said method comprising: administering to said subject a
pharmaceutical effective amount of said nutritional or food or
pharmaceutical.
14. Method of increasing body mass and thermogenesis in a subject
in need thereof with a nutritional or food or pharmaceutical
composition comprising the bacterial strain Lactobacillus paracasei
LMG S-26420, said method comprising: administering to said subject
a pharmaceutical effective amount of said nutritional or food or
pharmaceutical composition comprising the bacterial strain
Lactobacillus paracasei LMG S-26420.
Description
STATE OF THE ART
[0001] The present invention relates to a new strain belonging to
the species Lactobacillus paracasei, called LMG S-26420, able to
convert linoleic acid (LA) into conjugated linoleic acid (CLA),
useful in the treatment and prevention of diseases and/or
physiological states related to conjugated linoleic acid deficiency
or in cases where it is recommended the use of a probiotic, to
processes for obtaining it and to pharmaceutical or nutritional
compositions containing it.
[0002] The strain Lactobacillus paracasei LMG S-26420 is also
useful in the treatment and prevention of all disorders where the
assumption of a probiotic is beneficial, for example for restoring
and maintaining the intestinal flora balance.
[0003] The term CLA refers to a mixture of positional and
geometrical isomers of linoleic acid (LA), wherein the double bonds
are conjugated in various positions, thus generating cis- and
trans-isomers.
[0004] At least 16 CLA isomers are known and the isomers of major
biological interest are the isomers c9-t11 and t9-t11. The isomer
c9-t11 is the predominant isomer in diets, and it has proved to be
important because it is involved in many biological processes and
is incorporated in the phospholipidic fraction of animal tissues,
which are fed with mixtures of CLA isomers.
[0005] The CLA production in human organism is substantially
irrelevant and its supply is therefore entrusted to the ingestion
of dairy products and meat through diet or supplements containing
probiotic bacteria.
[0006] Foods produced by ruminants are the major CLA source for
humans. They are intermediates in the bio-hydrogenation of linoleic
acid and it is generally accepted that CLA in ruminants is
originated by the incomplete bio-hydrogenation of the unsaturated
acids of the linoleic acid by ruminal bacteria.
[0007] CLA is introduced in the organism through a diet with milk,
fish, meat and dairy products, and its beneficial effects are
correlated to daily assumption levels of about 3 g/day. An
unbalanced diet gives an average CLA assumption of 0.35 g/day and,
considered its beneficial effects for the organism, this deficiency
must be compensated through the assumption of probiotics converting
LA into CLA to provide useful dosages.
[0008] The biological activities of the conjugated linoleic acids
are largely attributed to the action of isomers c9-t11 and t10-c12;
the major biological activities linked to anti-carcinogenic effects
are attributed to the isomer c9-t11, while the isomer t10-c12 is
involved in the lipid metabolism of the human body.
[0009] CLA has important biological properties and is useful for
human and animal health, such as e.g. in intestinal inflammatory
pathologies, diarrhea and colon inflammation, in increasing body
mass, in increasing thermogenesis, in oxidative stress protection,
in tumour prevention, in autoimmune diseases, inflammatory
diseases, diabetes and atherosclerosis.
[0010] Ecker J. et al. in Biochem. Bioph. Res. Comm. 388, 2009,
660-666 report that the isomer c9-t11 activates the target gene LXR
involved in the development and progress of atherosclerosis. Ogawa
J. et al in Appl. Env. Microbiol 2001, 67, 1246 describe the
production of specific CLA isomers from linoleic acid by
lactobacilli, Lactobacillus acidophilus in micro-aerophilic
conditions, assuming that the hydroxy acid
10-hydroxy-cis-12-octadecanoid is the intermediate for this
conversion and that the conversion involves more than one step.
[0011] Ogawa J. et al. in J. Bioscience Bioeng. 100 (4), 355 (2005)
report the conversion of LA into CLA by bifidobacteria and
lactobacilli with the obtainment of a mixture of isomers. When
bifidobacteria are used, the production of CLA isomers varies from
about 3 to about 400 mg per litre of culture, while when
lactobacilli are used, the productivity varies from about 100 mg to
about 4 g per litre of culture. When Lactobacillus casei are used,
the productivity never exceeds 1 g per litre of culture.
[0012] Rosberg-Cody E. et al in Appl. Env, Microbiol 70 (8) 4635,
2004 describe the isolation of bifidobacteria strains from new-born
faecal material, useful for the production of CLA, and suggest the
use of those bacteria in supplements for new-borns at risk of
necrotizing enterocolitis.
[0013] Also Coakley M. et al. in J. Appl. Microbiol. 94, 138 (2003)
describe the ability of lactobacillus, lactococcus and
bifidobacteria to convert LA into CLA, and demonstrate that
bifidobacteria strains have a higher ability of conversion of LA
into CLA, with conversion percentages up to almost 65%.
[0014] Alonso L. et al. in J Diary Sci. 86, 1941 (2003) describe
the conversion of LA into CLA by Lactobacilli casei and
Lactobacilli acidophilus of human intestinal origin in media added
with different concentrations of linoleic acid. At concentrations
of 0.02% of linoleic acid, the maximum CLA concentration obtained
with Lactobacillus casei is about 110 mg per litre of culture and
the amount of the isomer of biological interest c9-t11 ranges from
60 to 85 mg per litre of culture.
[0015] Gustavo A. et al, in Biosci. Biotechnol. Biochem., 77 (3),
648-650, 2013, describe a rapid and simpler method to screen
conjugated linoleic acid (CLA)-producing bacteria isolated from cow
milk. One strain resembling a L. Paracasei converted free linoleic
acid to total CLA in a percentage greater than 85%. However the
percentage of conversion in CLA isomer of biological interest
c9,t11 is only about 18%.
[0016] Oguz Gursoy et al, in International Journal of Food Sciences
and Nutrition, 63(5), 610-615, describe the effect of using
different probiotics cultures on conjugated linoleic acid
concentration and fatty acid composition of cheese. They report
that the probiotic differences and storage process have not
affected the CLA contents of the samples statistically. The
increment of CLA content of cheese samples is due to lipolysis of
the free linoleic acid by lactic acid bacteria.
[0017] The assumption of probiotic products comprising lactobacilli
is also useful for maintaining the intestinal bacterial flora
balance and is a useful tool in the prevention and treatment of
dysbiosis in general, where the balance between bifidobacteria and
lactobacilli is essential.
[0018] Given the low amount of CLA introduced with the diet, and
the dosage useful in all pathologies and disorders connected to a
CLA deficiency, there was the need to have available bacteria
converting LA into CLA for obtaining daily dosages useful for the
health of individuals and for the treatment of all pathologies
where CLA isomers are beneficial.
[0019] Bifidobacteria convert LA into CLA, but bifidobacteria
cultures have the disadvantage of having low productivity yields,
meant as number of bacteria per litre of culture, and therefore it
is not easy to obtain them in large amounts in industrial scale
processes.
[0020] Therefore, there was the need to have a bacterial strain
able to convert LA into CLA obtainable by bacterial cultures with
high productivity to be used in pharmaceutical or nutritional
preparations useful in the treatment or prevention of all disorders
and pathologies related to a CLA deficiency.
[0021] There was also the need to have a bacterial strain belonging
to the genera Lactobacillus, able to convert LA into CLA isomers
beneficial for humans or animals. Moreover, there was also the need
to have a lactobacillus for all disorders or pathologies associated
with a microbial imbalance on the body surface. A strain belonging
to the lactic genus can be ingested in association with bacteria of
the genus Bifidus to promote the microbial balance of human or
animal flora.
[0022] The bacteria belonging to the genus Lactobacillus are among
those with the highest productivity; therefore, they are preferable
to those belonging to the genus Bifidobacterium to convert LA into
CLA. Lactobacilli can be obtained by industrial processes with
higher yields than bifidobacteria, and can be used for the
production of nutritional or pharmaceutical compositions for the
administration in human or animal.
[0023] Among these, lactobacilli able to convert LA into isomers
c9-t11 and t9-t11 of CLA to a greater extent than the other isomers
are preferred. c9-t11 is a major isomer because it is involved in
cell membrane phospholipids and is the predominant isomer in diets.
These isomers have beneficial effects on humans and animals, and in
particular the isomer t9-t11 has anti-proliferative and anticancer
properties. Ecker J et al. in Biochem. Biophysical. Res. Comm. 388,
660, 2009 report that the isomer t9-t11 of CLA is a powerful
agonist of macrophagic LXR, linked to the inflammation induction
processes, and it has an important role in the reduction of
arteriosclerotic processes in animal models.
[0024] The present invention describes a strain belonging to the
genus Lactobacillus, named Lactobacillus paracasei LMG S-26420,
able to convert LA into CLA with a conversion percentage higher
than the other lactobacilli strains known in the art. Moreover, the
strain LMG S-26420 of the present invention is characterized by
converting LA into CLA, wherein the mixture of isomers c9-t11 and
t10-c12 is prevalent if compared to the other isomers.
[0025] The strain is obtained with bacterial cultures characterized
by a productivity higher than 4 g per litre of culture and it can
be obtained in lyophilic form.
[0026] The strain is stable and the product of the bacterial
culture can be stored for long periods at temperatures lower than
0.degree. C. or for periods longer than 6 months at 4.degree. C. in
lyophilic form. The strain LMG S-26420 can be comprised in
nutritional or food preparations or in pharmaceutical compositions
useful for the treatment and/or the prevention of disorders or
pathologies linked to a CLA deficiency and in all disorders wherein
a probiotic assumption is beneficial for humans or animals.
SUMMARY
[0027] The present invention describes a new strain belonging to
the genus Lactobacillus, named Lactobacillus paracasei, filed at
the Belgian Coordinated Collections of Microorganisms BCCM/LMG
Bacteria Collection-Microbiology Laboratory-Ghent University on
Apr. 15, 2011 with the number LMG S-26420.
[0028] The Lactobacillus Paracasei LMG S-26420, is characterized in
that it converts linoleic acid in conjugated linoleic acid in a
percentage higher than 30%.
[0029] The strain Lactobacillus paracasei LMG S-26420 is
characterized by converting linoleic acid (LA) into conjugated
linoleic acid (CLA), in a percentage higher than 30% if compared to
the starting LA, wherein the biological isomers with biological
activity c9-t11 and t9-t11 are in a percentage higher than 30% if
compared to the other CLA isomers.
[0030] The strain Lactobacillus paracasei LMG S-26420 is
characterized by the production of high concentration of CLA.
[0031] The strain Lactobacillus paracasei LMG S-26420 is obtained
by bacterial cultures characterized by a productivity higher than 4
g per litre of culture and a yield of Colony Forming Units (CFU)
higher than 1.times.10.sup.9 per millilitre of culture.
[0032] The strain Lactobacillus paracasei LMG S-26420 is
characterized by being stable: it can be stored at temperatures
lower than 0.degree. C. and for periods longer than 6 months at
4.degree. C. in lyophilic form.
[0033] The strain lyophilic Lactobacillus paracasei LMG S-26420 is
characterized by an amount of living cells higher than
1.times.10.sup.10, in particular from about 1.times.10.sup.10 to
about 7.times.10.sup.10 unit per gram of lyophilic product.
[0034] It is an object of the present invention the process for the
production of the strain Lactobacillus paracasei LMG S-26420 in
bacterial culture, wherein the inoculum of the strain LMG S-26420
has a concentration from 0.1 to 10% (v/v) in a culture medium at a
temperature comprised between 30.degree. C. and 37.degree. C., at
pH values comprised between 4.5 and 7.5, for a period comprised
between 6 and 15 hours. The biomass is separated and it can be
preserved at temperatures lower than 4.degree. C. or submitted to
lyophilisation processes.
[0035] The described process leads to the obtainment of the strain
Lactobacillus paracasei LMG S-26420 with a number of colony-forming
units comprised between 1.times.10.sup.9 and 5.times.10.sup.9 CFU
per millilitre of culture and with a biomass in an amount comprised
between 10 and 20 grams per litre of culture.
[0036] The process comprising the lyophilisation leads to the
obtainment of the strain LMG S-26420 in solid form. In the presence
of cryoprotectants, the strain is obtained with yields of
colony-forming units (CFU) higher than 50% and the lyophilic strain
LMG S-26420 includes an amount of cells higher than
1.times.10.sup.10 per gram of lyophilic product.
[0037] Objects of the invention are nutritional or food
preparations and pharmaceutical compositions comprising an amount
of Lactobacillus paracasei LMG S-26420 in lyophilic form
corresponding to an amount of living cells from about
1.times.10.sup.8 to about 5.times.10.sub.11.
[0038] Nutritional or food preparations and pharmaceutical
compositions comprising Lactobacillus paracasei LMG S-26420 can be
in form of sachets, tablets or capsules. The nutritional or food
preparations and the pharmaceutical compositions can comprise
prebiotics, vitamins, mineral salts and pharmaceutical or
nutritional excipients.
[0039] The prebiotics are comprised in Lactobacillus paracasei LMG
S-26420 compositions, selected from the group consisting of
fructo-oligosaccharides, inulines, galacto-oligosaccharides,
xilo-oligosaccharides, isomalto-oligosaccharides and vitamins
selected from the group comprising the vitamins of the E and B
complex.
[0040] The nutritional or food preparations and pharmaceutical
compositions comprising Lactobacillus paracasei LMG S-26420 can
comprise bifidobacteria.
[0041] The compositions comprising Lactobacillus paracasei LMG
S-26420 are useful in the treatment and prevention of pathologies
and/or physiological states related to the deficiency of conjugated
linoleic acid and in all other cases wherein the use of a probiotic
is useful and has a beneficial effect.
[0042] In particular, the compositions comprising Lactobacillus
paracasei LMG S-26420 are useful in the treatment and prevention of
pathologies and/or physiological states related to a CLA
deficiency, such as e.g. in intestinal inflammatory pathologies, as
diarrhoeal and colon inflammation; in increasing body mass, in
increasing thermogenesis, in oxidative stress protection, in tumour
prevention, in autoimmune diseases, diabetes and
atherosclerosis.
[0043] The compositions comprising Lactobacillus paracasei LMG
S-26420 with bifidobacteria, object of the present invention, are
useful in the treatment of all disorders related to bacterial
dysbiosis.
[0044] The compositions comprising Lactobacillus paracasei LMG
S-26420 are useful in all physiological states where the intestinal
bacterial flora balance of humans and animals must be kept
unaltered.
DESCRIPTION OF THE INVENTION
[0045] A pure bacterial culture of the strain belonging to the
Lactobacillus paracasei was filed at the Belgian Coordinated
Collections of Microorganisms-BCCM/LMG Bacteria
Collection-Microbiology Laboratory-Ghent University with the number
LMG S-26420.
[0046] The present invention refers to a new strain of
Lactobacillus paracasei LMG S-26420, to pharmaceutical, nutritional
or food compositions comprising said strain and to their use in the
treatment and prevention of pathologies and/or physiological states
related to the deficiency of conjugated linoleic acid or when the
use of a probiotic is suggested.
[0047] The strain Lactobacillus paracasei LMG S-26420 has been
isolated from the vaginal bacterial flora of a healthy woman,
selected among many other strains of lactobacilli simultaneously
isolated from the same source and from other types of biological
samples and among other strains of the same species, because it has
shown an ability to convert linoleic acid (LA) into conjugated
linoleic acid (CLA). This characteristic makes this strain a
probiotic agent useful for human and animal use.
[0048] Lactobacillus paracasei LMG S-26420 is characterized by
converting LA into CLA in a percentage higher than 30% determined
by chromatographic method.
[0049] Lactobacillus paracasei LMG S-26420 converts LA into isomers
of CLA with biological activity, in particular in the isomers
c9-t11 and t9-t11. In a particular aspect of the invention,
Lactobacillus paracasei LMG S-26420 converts LA into the isomers
c9-t11 and t9-t11 of CLA in a percentage higher than 30%.
[0050] To evaluate the ability of the strain Lactobacillus
paracasei LMG S-26420 to convert LA into CLA, three different
methods were used: the Ogawa method, the Liu method and the
chromatographic method.
[0051] The use of the Ogawa J. method described in Appl. Environ.
Microbiol 67(3): 1246-1252, 2001 allows determining the ability of
the strain Lactobacillus paracasei LMG S-26420 to convert LA into
CLA in a suitable culture medium by adding LA to the bacterial
culture in order to metabolically adapt the cells.
[0052] In a first case, variable concentrations of LA, in a range
from 0.01 mg/ml to 1 mg/ml, are added to a series of cell cultures
of the strain LMG S-26420 and cells are incubated for a period from
1 to 4 days at temperatures from 30.degree. to 40.degree. C. The
final product is centrifuged and a constant concentration of LA,
equal to 5 mg/ml, was added to the cells. The cell cultures are
incubated at a temperature ranging from 30.degree. to 40.degree.
C., for a period from 40 to 80 hours and then centrifuged to remove
the supernatant. To evaluate the ability of a strain to convert LA
into CLA, the cells are resuspended in water and CLA is determined
by spectrophotometric method.
[0053] In a second case, a constant concentration of LA equal to 5
mg/ml is added to a series of cell cultures of Lactobacillus
paracasei LMG S-26420 and the biomass obtained at the end of the
culture is incubated with variable concentrations of LA in a range
from 0.05 mg/ml to 0.4 mg/ml.
[0054] In both cases, Lactobacillus paracasei LMG S-26420 shown to
be effective in converting LA into CLA, in particular in a
percentage higher than 30%.
[0055] The use of the Liu P. method as described in Biomed. &
Biotechnol. 12811, 923-930, 2011, comprises the inoculation of the
strain LMG S-26420 in an adequate culture medium with LA
concentrations from 0.05 to 1 mg/ml. The cultures are incubated for
a period from 1 to 3 days at temperatures from 30.degree. C. to
40.degree. C. and then centrifuged. The CLA concentrations is
determined by means of spectrophotometric method.
[0056] The trials carried out according to the Ogawa and Liu
methods showed the ability of the strain Lactobacillus paracasei
LMG S-26420 to convert LA into CLA. However, it is not possible to
evaluate the percentage of conversion into CLA isomers with these
methods, in particular if the strain is able to convert LA into CLA
isomers having the strongest biological activity.
[0057] The chromatographic method, in turn, allows the separation
and quantification of CLA geometrical isomers, isomers trans-trans,
trans-cis and cis-cis, in particular isomers cis9-trans 11, trans
10-cis 12 and trans 9-trans 11.
[0058] The obtained CLA isomers were determined by using silver-ion
high-pressure liquid chromatography (HPLC) with a diode array
detector and a UV detector at 234 nm.
[0059] The chromatographic method demonstrates that the strain LMG
S-26420 converts LA into CLA with a percentage higher than 30%.
[0060] The chromatographic method demonstrates that the strain LMG
S-26420 converts LA into CLA with a percentage from 30% to 50%,
wherein the percentage of isomers with biological activity, c9-t11
and t9-t11, are prevalent if compared to the other isomers. In
particular, the strain LMG S-26420 converts LA into CLA isomers
c9-t11 and t9-t11, wherein their percentage is 40% higher than all
other CLA geometrical isomers.
[0061] The strain LMG S-26420 is obtained by cell cultures
characterized by giving a number of colony forming units (CFU)
higher than 1.times.10.sup.9 per millilitre and an amount of solid
mass higher than 4 g per litre of culture. The obtained new
bacterial strain is stable at temperatures lower than 4.degree. C.
for longer periods and it can be lyophilised with processes that
significantly preserve cell viability. The lyophilic products are
stable for periods of time longer than 3 months at temperatures of
4.degree. C. and 25.degree. C.
[0062] The strain Lactobacillus paracasei LMG S-26420 produces an
amount of lyophilised biomass higher than 4 g per litre of culture,
in particular from 10 to 20 g per litre of culture. Considering the
ability of this strain to convert LA into CLA in a percentage
higher than 30%, it can be stated that the new strain Lactobacillus
paracasei LMG S-26420 is able to produce remarkable concentrations
of CLA. For example, adding Lactobacillus paracasei LMG S-26420 to
solutions including concentrations of about 500 mg/litre of
linoleic acid leads to obtain concentrations higher than 250
mg/litre of conjugated linoleic acid.
[0063] Another advantage of the present invention is to have a new
strain belonging to the genus Lactobacillus for the conversion of
LA into CLA isomers with high conversion yields useful to be
included in nutritional or food preparations or in pharmaceutical
compositions.
[0064] The new strain Lactobacillus paracasei LMG S-26420 can be
comprised in compositions also comprising other bacterial strains,
belonging to the same genus or not, like bifidobacteria.
[0065] The compositions comprising LMG S-26420 and other bacterial
strains advantageously generate a heterogeneous probiotic
population, useful to maintain the intestinal bacterial flora
balance.
[0066] The bacterial cultures object of the present invention were
produced first in laboratory scale and then in industrial scale.
The bacterial culture of LMG S-26420 was obtained by means of a
fermentation process in a period from 6 to 12 hours at temperatures
ranging from 30.degree. C. to 40.degree. C. in a medium named
MRS.RTM. (De Man, Rogosa & Sharpe) containing as main
ingredients yeast extract, mixtures of peptones and glucose in
addition to potassium salts, ammonium, magnesium and manganese.
[0067] Starting from the primary cultures, many expansion phases
were conducted to increase the number of cells per volume of
culture of the pure strain in order to obtain the so called "mother
cultures", that were used as inoculum for the industrial production
of the bacterial cultures of the probiotic LMG S-26420.
[0068] The industrial process for the production of Lactobacillus
paracasei LMG S-26420 comprises the following steps: [0069]
inoculating the strain LMG S-26420 with volumes of mother cultures,
in a volumetric percentage from 0.1 to 10% and fermenting in an
adequate medium of culture at 37.degree. C. and pH from 4.5 to 7.5,
for a period from 8 to 15 hours; [0070] separating the bacterial
biomass from the culture broth by centrifugation.
[0071] The biomass can be frozen or submitted to lyophilisation
processes after adding suitable cryoprotectants, selected among
soluble carbohydrates, which are useful to maintain the cell
viability during the lyophilisation processes.
[0072] The strain LMG S-26420 is obtained by fermentation processes
characterized by producing a number of living cells higher than
10.sup.9 living cells per ml of culture and a lactobacilli biomass
higher than 4 g per litre of culture.
[0073] The strain LMG S-26420 is obtained with a productivity from
1.times.10.sup.9 to 7.times.10.sup.9 living cells per ml of culture
and has a yield of dried product from 10 to 20 g per litre.
[0074] The cell viability is determined using bacterial counting
methods known to the person skilled in the art.
[0075] The strain LMG S-26420 obtained from the described cultures
can be lyophilised to be easily stored and to be included in
alimentary, food or pharmaceutical preparations.
[0076] The lyophilisation process was executed in the presence of
cryoprotectants chosen among soluble carbohydrates, such as for
example trealose and cyclodextrins or a mixture of them. The
described lyophilisation process is characterized by obtaining a
lyophilic strain LMG S-26420 with a yield in vital cells higher
than 50% than before the lyophilisation process. The strain LMG
S-26420 is characterized by comprising an amount of living cells
per gram of lyophilic product higher than almost 1.times.10.sup.10,
in particular from almost 1.times.10.sup.10 to almost
7.times.10.sup.10 per gram of lyophilic product. This confirms that
the strain Lactobacillus paracasei LMG S-26420 is characterized by
a high productivity, can be lyophilised and maintained as lyophilic
product with the certainty of preserving cell viability.
[0077] The strain Lactobacillus paracasei LMG S-26420 obtained with
the described cell cultures and lyophilised in presence of
cryoprotectants selected among soluble carbohydrates or mixtures
thereof, is characterized by a water activity (a.sub.w) lower than
0.6, a value below which the proliferation of the majority of
bacteria and mildews is inhibited. The water activity is a value
obtained by measuring the partial steam pressure in a substance
divided by the partial pressure of water, and is obtainable with
detectors directly giving those values.
[0078] As described by Ryser, E. T. et al in Listeria, Listeriosis
and Food Safety (3rd ed.). CRC Press. 173-174, (2007), water
activity values lower than 0.6 assure that the lyophilic
preparation can be stored because it does not degrade because of
microbial proliferation, which is one of the most dangerous causes
of alimentary or food alterations.
[0079] The lyophilic products of the strain LMG S-26420s are
characterized by having an elevated number of living cells per gram
of lyophilic product and a low content of water activity, are
useful to be stored for the preparation of compositions or
preparations in different forms containing different probiotic
amounts, with no limitation.
[0080] The LMG S-26420 bacterial strain in the solutions submitted
to the lyophilisation processes is characterized by values of glass
transition temperatures, T.sub.g, higher than 100.degree. C., and
in the presence of soluble carbohydrates, such as e.g. trealose,
the values of glass transition temperatures are in a range from
100.degree. C. to 120.degree. C. These values confirm that this
strain can be submitted to lyophilisation and vacuum drying
processes up to temperatures of 100.degree. C. without having the
product converted into a glassy state, with a consequent loss of
its properties.
[0081] The glass transition temperature of frozen solutions
T.sub.g' relating to the preparations to be lyophilised is in a
range from -15.degree. to -30.degree. C. and in the presence of
soluble carbohydrates, as e.g. trealose, the glass transition
temperatures are in a range from -20 to -30.degree. C.
[0082] The lyophilisation process of the strain Lactobacillus
paracasei LMG S-26420 described in Example 3 in the presence of
cryoprotectants chosen among soluble carbohydrates, in particular
trealose, is characterized by a freezing step carried out at
temperatures lower than -30.degree. C. and a secondary drying
carried out at temperatures lower than 100.degree. C. Said process
leads to the obtainment of the strain LMG S-26420 in lyophilic
form, which is characterized by maintaining its biological
properties.
[0083] In particular, the lyophilisation process of the strain
Lactobacillus paracasei LMG S-26420 described in the invention in
the presence of trealose maintains a cell viability higher than
30%, in particular 30% to 60%, if compared to the cell viability
before lyophilisation.
[0084] The described lyophilic compositions of the strain
Lactobacillus paracasei LMG S-26420 comprise soluble carbohydrates,
such as trealose and mannitol; this latter can be added to
trealose-comprising lyophilic preparations when a high lyophilic
mass is requested, in particular to be used in preparations with a
low dosage of probiotic strain.
[0085] The solutions comprising Lactobacillus paracasei LMG S-26420
to be lyophilised can comprise osmolytes chosen from the group
consisting of betaine, sarcosine, glycerol, erythritol; salts
selected from the group consisting of acetates, formiates or
ammonium salts, useful to maintain pH values between 4 and 8 during
the lyophilisation process.
[0086] The lyophilic strain Lactobacillus paracasei LMG S-26420 is
stable at a temperature of 4.degree. C. for a period longer than 3
months. In a particular aspect, Lactobacillus paracasei LMG S-26420
maintains more than 70% of its cell viability after 6 months at the
temperature of 4.degree. C., and the water activity remains with
values lower than 0.6%.
[0087] The lyophilic product can therefore be prepared in large
amounts and be stored for the preparation of compositions in solid
or suspended form at different probiotic dosages.
[0088] An aspect of the present invention refers to pharmaceutical
compositions, nutritional or food preparations comprising variable
amounts of the strain LMG S-26420 in lyophilic form in amounts
ranging from 20 to 2500 mg. These compositions are characterized by
comprising an amount of living cells of LMG S-26420 from
1.times.10.sup.9 to 1.times.10.sup.11 per gram of lyophilic
product.
[0089] The preparations can be in a form useful for oral
administration, such as e.g. in sachets, tablets, capsules or
liquid suspensions.
[0090] The compositions in form of tablets or capsules can comprise
an amount of the strain Lactobacillus paracasei LMG S-26420 ranging
from 20 to 800 mg and the sachet compositions for liquid suspension
can comprise an amount of Lactobacillus paracasei LMG S-26420
ranging from 20 mg to 10 grams. The pharmaceutical or nutritional
compositions can comprise an amount of Lactobacillus paracasei LMG
S-26420 ranging from about 1.times.10.sup.9 to 1.times.10.sup.11
unity of cells.
[0091] The pharmaceutical, nutritional or food compositions
comprising Lactobacillus paracasei LMG S-26420 can optionally
comprise, but are not limited to, prebiotics selected from the
group consisting of fructo-oligosaccharides, inulines,
galacto-oligosaccharides, xilo-oligosaccharides,
isomalto-oligosaccharides, resistant dextrin, polydextrose,
arabinogalactans, resistant starch, dextrans, guar gum; amino
acids; proteins; antioxidants; vitamins selected from the group
comprising the vitamins of the E and B complex, together with
pharmaceutically acceptable salts useful for the preparation of the
desired form.
[0092] The pharmaceutical, nutritional or food compositions
comprising Lactobacillus paracasei LMG S-26420 can comprise also
other bacteria able to convert LA into CLA, in particular those
belonging to the genus Bifidobacterium. These compositions increase
the conversion of LA into CLA and favour the intestinal bacterial
flora balance.
[0093] The compositions in form of sachets are prepared by mixing
the strain Lactobacillus paracasei LMG S-26420 in form of lyophilic
products with selected excipients, such as e.g. oligosaccharides,
selected from the group of fructo-oligosaccharides, inulines,
galacto-oligosaccharides, xilo-oligosaccharides,
isomalto-oligosaccharides and flavours, previously sieved. The
homogeneous mixture is then divided in sachets.
[0094] The lyophilic form can be grinded or granulated before being
added to the pharmaceutical excipients chosen for the preparation
of the desired solid forms.
[0095] The compositions in form of tablets can comprise diluents,
ligands, disintegrants, lubricants, glidants and are prepared
according to the techniques known to the person skilled in the
art.
[0096] Optionally, the compositions can comprise preservatives,
antioxidants, buffering, colouring, flavouring and sweeting
agents.
[0097] The compositions of the invention comprising the strain
Lactobacillus paracasei LMG S-26420 are stable at temperature of
4.degree. C. and 25.degree. C. for a period of 1, 3 and 6 months
with a complete recovery of the living cells.
[0098] Another aspect of the invention refers to the use of
pharmaceutical, nutritional or food compositions comprising the
strain Lactobacillus paracasei LMG S-26420 for use in the treatment
and prevention of pathologies and/or physiological states wherein a
probiotic is useful. These compositions are useful in the
prevention and treatment of pathologies and/or physiological states
related to a CLA deficiency such as, e.g. in intestinal
inflammatory pathologies, diarrhea and colon inflammation, in
increasing body mass, in increasing thermogenesis, in oxidative
stress protection, in tumour prevention, in autoimmune diseases,
inflammatory diseases, diabetes and atherosclerosis.
[0099] The compositions comprising the strain Lactobacillus
paracasei LMG S-26420 are useful in the treatment and prevention of
all disorders or pathologies connected to a microbial
unbalance.
[0100] The compositions comprising the strain Lactobacillus
paracasei LMG S-26420 are also useful as alimentary supplement in
all diets poor of meat or milk products to obtain CLA
concentrations beneficial for humans.
EXAMPLES
[0101] The Examples refer to the strain Lactobacillus paracasei LMG
S-26420 exactly corresponding to the one filed at the Belgian
Coordinated Collection of Microorganisms, BCCM, LMG Bacteria
Collection, Microbiology Laboratory, Ghent University, which
confirmed the purity and viability of the strain and registered it
with the number LMG S-26420. In particular, the examples refer to
the characterization of the strain LMG S-26420, to its ability to
convert LA into CLA, to its production process in industrial scale
and to compositions containing it.
Example 1
[0102] Determination of the Strain Ability to Convert Linoleic Acid
(LA) into Conjugated Linoleic Acid (CLA)
[0103] To determine the ability of the strain Lactobacillus
paracasei LMG S-26420 to convert LA into CLA, it has been evaluated
a condition wherein the inoculum was made in presence of LA, to
stimulate bacterial cells pre-adaptation, and a condition without
LA.
[0104] a) Determination According to the Ogawa Method
[0105] The quantitative determination of the ability of
Lactobacillus paracasei LMG S-26420 to convert LA into CLA was
carried out according to the Ogawa method described in Appl. Envr.
Microbiol. 2001, 67, 1246.
[0106] The method is based on the inoculum of the strain in
MRS.RTM. (Man, Rogosa, Sharpe) cultural medium comprising a mixture
of peptones 18 g/l; yeast extract 4 g/l; glucose 20 g/l; tween-80 1
ml/l; potassium phosphate 2 g/l; tri-ammonium citrate 2 g/l;
anhydrous sodium acetate 3 g/l; heptahydrate magnesium sulphate 0.2
g/l; anhydrous magnesium sulphate 0.034 g/l; agar 12 g/l.
[0107] The strain Lactobacillus paracasei LMG S-26420 at a
concentration of 1% was inoculated in 15 ml of MRS.RTM. with LA at
concentrations from 0.01 to 0.4 mg/ml and the solutions were kept
at a temperature of 37.degree. C. for 3 days at low stirring. The
cultures were centrifuged and the supernatant was eliminated. The
pellet was washed with sterile water and the cell mass of about 20
mg was resuspended with 1 ml of solution of potassium phosphate
buffer 100 mM at pH 6.5 and 5 mg of LA in a complex with bovine
albumin (BSA) were added to cell suspension, with a ratio
corresponding to 0.2 mg BSA/mg of LA. The cultures were then
incubated for 48 and 72 hours and then centrifuged to eliminate the
supernatant. The cells were resuspended in water and the CLA
concentration was spectrophotometrically determined by means of the
Barret method, described in Appl. Environment Microbiol. 73(7),
2333, (2007). This method comprises the extraction of the fatty
acid fraction by the addition of 2 ml of isopropanol to 1 ml of
sample. The solutions were vigorously stirred, allowed to stand for
3 minutes and then 1.5 ml of hexane were added. Organic phases were
separated and dehydrated with anhydrous sodium sulphate and the CLA
amount was determined by spectrophotometric reading at 233 nm. The
concentrations were determined by a calibration curve obtained with
different concentrations of the CLA isomer c9-t11 at the same
wavelength.
[0108] Table 1 reports the conversion percentages of LA into CLA by
means of the strain Lactobacillus paracasei LMG S-26420, wherein
the pre-incubation of the strain was carried out at different
concentrations of LA and the incubation of the washed cells was
carried out with 5 mg/ml of LA. Table 1 shows that, in the reported
conditions, the conversion percentages of LA into CLA range from 1%
to 7%.
TABLE-US-00001 TABLE 1 Cell Conversion LA Cell incubation CLA
percentage concentration incubation temperature concentration (%)
of LA (mg/ml) time (h) (.degree. C.) (mg/ml) into CLA 0.01 48 30
0.083 2 0.05 48 30 0.075 2 0.1 48 30 0.067 1 0.2 48 30 0.041 1 0.3
48 30 0.045 1 0.4 48 30 0.104 2 0.01 72 37 0.204 4 0.05 72 37 0.273
5 0.1 72 37 0.097 2 0.2 72 37 0.349 7 0.3 72 37 0.282 6 0.4 72 37
0.085 6
[0109] Under the conditions reported in Table 2, CLA is not present
in the supernatant.
[0110] Table 2 reports the results of the conversion of LA into
CLA. The pre-incubation was carried out with LA at a concentration
of 0.05 mg/ml and the washed cell mass was incubated at a variable
concentration of LA. CLA was determined on the supernatant and on
the cell pellet.
TABLE-US-00002 TABLE 2 LA Cell CLA Conversion concen- Cell
incubation concen- percentage tration incubation temperature
tration (%) of LA (mg/ml) time (h) (.degree. C.) Sample (mg/ml)
into CLA 0.05 72 37 Cells -- -- 0.05 72 37 Supernat. 0.013 25 0.1
72 37 Cells -- 0.1 72 37 Supernat. 0.004 4 0.2 72 37 Cells -- 0.2
72 37 Supernat. -- -- 0.3 72 37 Cells -- -- 0.3 72 37 Supernat. --
-- 0.4 72 37 Cells 0.007 2 0.4 72 37 Supernat. 0.005 1 0.5 72 37
Cells 0.007 1 0.5 72 37 Supernat. 0.003 1 1 72 37 Cells 0.025 3 1
72 37 Supernat. -- -- 2 72 37 Cells 0.042 2 2 72 37 Supernat. 0.011
1 3 72 37 Cells 0.059 2 3 72 37 Supernat. 0.017 1 4 72 37 Cells
0.077 2 4 72 37 Supernat. 0.0043 1
[0111] b) Determination According to the Liu Method
[0112] The Liu method has been described in Biomed. Biotechnol.
12811, 923 (2011).
[0113] Table 3 reports data related to the conversion of LA into
CLA by Lactobacillus paracasei LMG S-26420, according to the method
described by Liu P et al. in Biomed. Biotechnol. 12811, 923,
2011.
[0114] The method is based on the determination of the CLA amount
in the supernatant of cultures in MRS.RTM. medium containing
different concentrations of LA from 0.05 to 0.5 mg/ml. The cultures
were incubated for a period of 24 and 96 hours and at temperature
of 30.degree. and 37.degree. C. The CLA obtained by the conversion
of LA was determined with the Barret method as described in the
Example 1.a.
TABLE-US-00003 TABLE 3 LA Cell CLA Conversion concen- Cell
incubation concen- percentage tration incubation temperature
tration (%) of LA (mg/ml) time (h) (.degree. C.) (mg/ml) into CLA
0.00 24 30 0.001 -- 0.05 24 30 0.005 9 0.1 24 30 0.005 5 0.2 24 30
0.005 2 0.3 24 30 0.006 2 0.4 24 30 0.006 2 0.5 24 30 0.006 1 0.00
96 30 0.001 -- 0.05 96 30 0.006 12 0.1 96 30 0.005 5 0.2 96 30
0.005 3 0.3 96 30 0.006 2 0.4 96 30 0.006 1 0.5 96 30 0.006 1 0.00
24 37 0.001 -- 0.05 24 37 0.004 8 0.1 24 37 0.005 5 0.2 24 37 0.005
2 0.3 24 37 0.005 2 0.4 24 37 0.006 1 0.5 24 37 0.006 1 0.00 96 37
0.001 -- 0.05 96 37 0.005 10 0.1 96 37 0.006 6 0.2 96 37 0.006 3
0.3 96 37 0.007 2 0.4 96 37 0.006 2 0.5 96 37 0.008 2
[0115] c) Determination According to the Chromatographic Method
(HPLC)
[0116] The chromatographic method allows the determination of CLA
geometrical isomers, the isomer c9-t11, the isomer trans 10-cis12
and the isomer trans9-trans11 in cell suspensions and in
supernatants of cultures according to the method described in a)
and in b) and reported in Tables 2 and 3.
[0117] The samples were methylated as described by Kramer J. et al.
in Am. J. Clin. 79, 1137 S 2004 and the obtained methyl esters were
separated by silver-ion high-pressure chromatography (HPLC). CLA
isomers were separated using three 4.6 mm.times.250 mm CHromSpher
Lipid columns, connected in series, with 5.mu.-sized particles. The
isomers were eluted with a solution of hexane-acetonitrile: 99-1
and detected with a diode array detector and a UV detector at 234
nm in series. The quantitative determination was carried out by
using solutions at known concentrations of CLA isomers.
[0118] Table 4a reports the determination of CLA isomers in pellet
samples coming from cultures with LA pre-incubation at a
concentration of 0.05 mg/ml and incubation of washed cells at
different concentrations of LA at 37.degree. C. and an incubation
time of 72 hours as reported in a), Ogawa method.
TABLE-US-00004 TABLE 4a CLA CLA CLA Isomers Total isomer isomer
isomer (t9-t11 and conversion LA conc. t9-t11 c10-t12 c9-t11
c9-t11)* percentage (mg/ml) (mcg/g) (mcg/g) (mcg/g) (%) (%) 0.05
1.39 2.75 2.07 3.46 14.8 0.01 1.56 3.50 3.07 4.63 0.2 0.4 1.21 2.78
2.40 3.61 0.2 *isomers with biological activity
[0119] Table 4b reports the concentration of CLA isomers in
supernatant samples coming from cultures incubated at different
concentrations of LA as reported in b), Liu method.
TABLE-US-00005 TABLE 4b CLA isomer Isomers Total CLA isomer CLA
isomer CLA isomer (t9-t11 (t9-t11 conversion LA conc. t9-t11
c10-t12 c9-t11 and c9-t11)* and c9-t11)* percentage (mg/ml) (mcg/g)
(mcg/g) (mcg/g) (mcg/g) (%) (%) 0.05 (24 h) 1.81 5.07 3.30 44.16
46.76 25.9 0.05 (96 h) 2.45 6.33 5.06 42.26 46.67 35.5 0.2 (96 h)
1.74 5.64 5.04 46.15 46.15 7.3 0.5 (96 h) 1.95 5.80 5.22 7.17 44.67
3.2 *isomers with biological activity
Example 2
[0120] Fermentation Process for the Preparation of the Strain
Lactobacillus paracasei LMG S-26420.
[0121] The fermentations were carried out in a Sartorius fermenter.
The inoculum of the strain Lactobacillus paracasei LMG S-26420 at
1%, corresponding to 40 ml, was cultured in 4 litres of MRS.RTM.
medium at a temperature of 37.degree. C. for 8 hours.
[0122] The cell mass obtained by different fermentations was
concentrated by centrifugation and washed with sterile water. The
optic density on the aqueous suspension of the cell mass was
determined by spectrophotometric method at 625 nm and the Colony
Forming Units (CFU) were determined by decimal plate count.
[0123] Some preparations were carried out with different
fermentation parameters in order to determine the best bacterial
growing conditions for the strain Lactobacillus paracasei LMG
S-26420.
[0124] Table 5 reports the parameters related to fermentations at
different carbon dioxide pressures, stirring speed and pH. Table 5
also reports the results obtained by measuring the optic density
and the CFU number on biomass at fermentation end, after 8
hours.
TABLE-US-00006 TABLE 5 Fermentation parameters Prep. 1 Prep. 2
Prep. 3 Prep. 4 Prep. 5 Prep. 6 Culture medium MRS .RTM. MRS .RTM.
MRS .RTM. MRS .RTM. MRS .RTM. MRS .RTM. + Trealose 1% pH n.c. n.c.
n.c. 5.5 5.5 5.5 P-CO2 (bar) 0.8 bar - 0.8 bar - 1 bar - 1 bar -
n.c. 0.8 bar - 30 min. 30 min. 60 min. 60 min. 45 min. Stirring
speed (rpm) 100 150 150 150 150 150 Biomass weight (g) 40 40 32 35
47 62 OD 625 nm - T0 3.50 0.075 0.041 0.045 0.036 0.04 OD 625 nm -
8 ore 0.087 1.78 1.406 1.63 1.553 1.50 CFU/ml - T0 3.50E+06
2.50E+07 6.30E+07 3.30E+07 3.00E+07 1.75E+07 CFU/ml -8 ore 2.30E+09
1.10E+09 1.90E+09 2.20E+09 2.60E+09 1.05E+09 n.c.: non
controlled
[0125] The CFU number of Lactobacillus paracasei LMG S-26420 at the
end of the culture was comprised in a range from 1.9.times.10.sup.9
to 2.6.times.10.sup.9 per millilitre of culture and the biomass
produced was comprised from 8 to 16 grams per litre of culture.
[0126] The CFU measure was carried out on Petri dish by serial
dilution plating of cell mass and microbial colony count.
[0127] Cell masses can be maintained at temperatures lower than
0.degree. C. or directly lyophilised to be preserved in solid form
or used for pharmaceutical/nutritional preparations.
Example 3
[0128] Preparation of the Strain Lactobacillus paracasei LMG
S-26420 in Lyophilic Form.
[0129] The product obtained from the preparation described in
Example 2 (Preparation 6) was lyophilised.
[0130] To value the effect of a cryoprotectant on the
lyophilisation of Lactobacillus paracasei LMG S-26420, the
lyophilisation processes were carried out in the presence of
cyclodextrins, trealose and mannitol.
[0131] The cell pellet was suspended in water and an amount of
cyclodextrins at 10% (p/V) was added.
[0132] The solution was divided in three parts:
[0133] Solution A: cyclodextrins 10% (w/V)
[0134] Solution B: cyclodextrins 10% (w/V)+trealose 20% (w/V)
[0135] Solution C: cyclodextrins 10% (w/V)+mannitol 15% (w/V).
[0136] Table 6 reports the lyophilisation process parameters for
the solutions A and B.
TABLE-US-00007 TABLE 6 STEPS of lyophilisation process (I)
Parameters Freezing -50.degree. C., v = 2.degree. C./min,
-50.degree. C. .times. 120 min Primary drying -50.degree. C.
.times. 15 min, -30.degree. C., v = 0.3.degree. C./min, -30.degree.
C. .times. 300 min P = 100 mTorr Secondary drying 30.degree. C., v
= 0.16.degree. C./min, 30.degree. C. .times. 480 min P = 100 mTorr
Total Time 14 hours
[0137] Table 7 reports the parameter of the lyophilisation process
for the solutions C.
TABLE-US-00008 TABLE 7 STEPS of lyophilisation process (I)
Parameters Freezing -60.degree. C. .times. 120 min Annealing
-10.degree. C., v = 0.55.degree. C./min -10.degree. C. .times. 240
min -50.degree. C. .times. 120 min Primary drying -18.degree. C., v
= 0.16.degree. C./min -18.degree. C. .times. 600 min P = 120 mTorr
Secondary drying 25.degree. C., v = 0.07.degree. C./min P = 50
mTorr
[0138] The obtained lyophilic strain Lactobacillus paracasei LMG
S-26420 was stored in bottles or sachets at 4.degree. C.
Example 4
[0139] Determination of Glass Transition Temperatures Tg, T'g The
glass transition temperature Tg and T'g of the solutions A and B
before lyophilisation was determined by differential scanning
calorimeter, using a Diamond DSC instrument, applying cycles of
freezing/heating as reported in Table 8.
TABLE-US-00009 TABLE 8 Temperature 1 Temperature 2 Speed Step
(.degree. C.) (.degree. C.) (.degree. C./min) 1 25 -60 10 2 -60 25
40 3 25 100 10 4 100 -50 50 5 -50 170 40
[0140] The cycle reported in Table 9 was used for the solutions
C.
TABLE-US-00010 TABLE 9 Temperature 1 Temperature 2 Speed Step
(.degree. C.) (.degree. C.) (.degree. C./min) 1 25 -60 10 2 -60 -10
40 3 -10 -10 Constant for 120 min 4 -10 -60 40 5 -60 25 40
[0141] Glass transition temperatures values of solutions Tg and of
frozen aqueous solutions, T'g of the solutions A, B and C to be
lyophilised are reported in Table 10.
TABLE-US-00011 TABLE 10 Lyophilisation T'g Tg LMG S-26420
Preparation (.degree. C.) (.degree. C.) Solutions A = cyclodextrins
A-1 -12.25 148.55 10% (p/V) A-2 -12.75 140.81 A-3 -13.05 144.44 B =
cyclodextrins 10% B-1 -26.50 112.00 (p/V) + Trealose 20% (pN) B-2
-26.63 114.64 B-3 -26.54 117.54 B-4 -26.75 112.45 B-5 -26.54 114.40
B-6 -30.58 111.93 B-7 -34.76 116.629 B = cyclodextrins 10% B-8
-27.96 111.77 (p/V) + Trealose 20% (pN) B-9 -27.50 118.40 C =
cyclodextrins 10% C-1 T'g.sup.1: -36.8 n.d. (p/V) + mannitol 15%
T'g.sup.2: -28 T'g.sup.3: -14.8 C-2 -28.4 106.00
Example 5
[0142] Characterization of the Lyophilic Strain Lactobacillus
paracasei LMG S-26420.
[0143] a) Strain Viability Determination
[0144] To determine the yield and viability of the strain
Lactobacillus paracasei LMG S-26420 after the lyophilisation
process of the solutions A, B and C prepared according to Example
3, the CFUs of the strain were determined before and after the
lyophilisation process, by Petri dish count. The determination was
made on an amount of lyophilic product corresponding to 0.5 grams,
suspended in 49.5 grams of dilution buffer (MRS.RTM.) and diluted
as serial dilution until reaching those useful for the count. The
diluted product was then deposited on a Petri dish containing 20 mL
of MRS.RTM. agar medium, previously sterilized in autoclave, and
incubated at 37.degree. C. in anaerobiosis for 72 hours.
[0145] The determination was carried out on many lyophilic
preparations made from the solutions A, B and C and the results are
reported in Table 11.
TABLE-US-00012 TABLE 11 Yield Lyo- percentage philisation Prep- (%)
lyo- LMG ara- Before-lyo After-lyo (T.sub.0) philisation S-26420
tion UFC/ml .+-. SD UFC/g .+-. SD process Solution A = A-1 4.6 .+-.
0.6 .times. 10.sup.10 5.6 .+-. 0.4 .times. 10.sup.10 13.9 .+-. 0.9
cyclodextrins A-2 4.0 .+-. 0.5 .times. 10.sup.10 3.4 .+-. 0.2
.times. 10.sup.10 16.6 .+-. 1.1 10% (wN) A-3 2.8 .+-. 0.5 .times.
10.sup.10 3.3 .+-. 0.5 .times. 10.sup.10 15.8 .+-. 2.9 B = B-1 3.2
.+-. 0.5 .times. 10.sup.10 2.9 .+-. 0.7 .times. 10.sup.10 26.2 .+-.
6.5 cyclodextrins B-2 4.4 .+-. 0.1 .times. 10.sup.10 3.3 .+-. 0.3
.times. 10.sup.10 22.8 .+-. 1.9 10% (wN) + B-3 3.3 .+-. 0.1 .times.
10.sup.10 6.3 .+-. 0.3 .times. 10.sup.10 57 .+-. 27.0 Trealose B-4
2.2 .+-. 0.8 .times. 10.sup.10 3.9 .+-. 0.3 .times. 10.sup.10 45.1
.+-. 2.7 20% (wN) B-5 2.8 .+-. 0.2 .times. 10.sup.10 4.6 .+-. 0.9
.times. 10.sup.10 41.7 .+-. 7.5 B-6 3.6 .+-. 0.1 .times. 10.sup.10
3.7 .+-. 0.5 .times. 10.sup.10 23.3 .+-. 2.9 B-7 3.1 .+-. 0.9
.times. 10.sup.10 2.9 .+-. 0.8 .times. 10.sup.10 21.9 .+-. 6.0 B-8
5.5 .+-. 0.7 .times. 10.sup.10 1.4 .+-. 0.8 .times. 10.sup.10 35.8
.+-. 1.4 B-9 4.3 .+-. 0.9 .times. 10.sup.10 3.7 .+-. 0.8 .times.
10.sup.10 22.6 .+-. 2.1 C = C-1 7.3 .+-. 2. .times. 10.sup.10 <1
.times. 10.sup.10 13.9 .+-. 0.9 cyclodextrins C-2 3.6 .+-. 0.5
.times. 10.sup.10 2.3 .+-. 0.8 .times. 10.sup.10 9.56 .+-. 4.0 10%
(p/V) + mannitol 15%
[0146] Table 11 confirms the effect of trealose during the
lyophilisation process, leading to a recovery higher than 30% in
CFU.
[0147] Mannitol can be added to trealose if a bigger lyophilic mass
is desired, in particular for small dosages of Lactobacillus
paracasei LMG S-26420.
Example 6
[0148] Determination of Water Activity and Weight Loss by Drying
Lyophilic Lactobacillus paracasei LMG S-26420.
[0149] a) Determination of the Water Activity (a.sub.w)
[0150] The determination of the water activity corresponds to the
determination of free water in lyophilic preparations of
Lactobacillus paracasei LMG S-26420 of the solutions A, B and C
prepared following Example 3. The determination was carried out by
placing a sample of about 1 gram of each lyophilic product in an
Aqualab VSA Decagon instrument that measures water activity of a
sample with a humidity dielectric detector, directly giving the
measure that is directly proportional to the water moles over the
sample moles and the obtained values are reported in Table 12.
TABLE-US-00013 TABLE 12 Lyophilisation components Preparation
a.sub.w LMG S-26420 No. (water activity) Solution A = cyclodextrins
A-1 n.d 10% (wN) A-2 n.d. A-3 0.358 B = cyclodextrins 10% (wN) +
B-1 n.d Trealose 20% (wN) B-2 n.d B-3 0.363 B-4 0.153 B-5 0.100 B =
cyclodextrins 10% (w/V) + B-6 0.120 Trealose 20% (w/V) B-7 0.136
B-8 0.166 B-9 0.087 Solution C = cyclodextrins 10% C-1 0.174 (w/V)
+ mannitol 15% (w/V) C-2 0.134
[0151] All obtained lyophilic have a.sub.w values lower than 0.6
confirming that they can be stored for long periods without risks
of degradation.
[0152] b) Determination of the Loss of Water Content (LOD)
[0153] The humidity content of the lyophilic samples was obtained
using a thermoscales Mettler and values are reported in Table
13.
TABLE-US-00014 TABLE 13 Humidity Lyophilisation components
Preparation Percentage LMG S-26420 No. LOD (%) Solution A =
cyclodextrins A-1 3.61 10% (w/V) A-2 2.58 A-3 3.9 B = cyclodextrins
10% (wN) + B-1 4.01 Trealose 20% (wN) B-2 4.24 B = cyclodextrins
10% (wN) + B-3 4.90 Trealose 20% (wN) B-4 3.39 B-5 2.94 B-6 2.85
B-7 3.05 B-8 3.21 B-9 2.41 Solution C = cyclodextrins 10% C-1 2.14
(w/V) + mannitol 15% (wN)
Example 7
[0154] Determination of Stability of Lyophilic Preparations of the
Strain Lactobacillus paracasei LMG S-26420.
[0155] The lyophilic preparations of the strain Lactobacillus
paracasei LMG S-26420 were placed at 4.degree. C. and their
stability was determined by measuring the bacterial charge and the
water activity (a.sub.w) (TO), at time zero, 3 and 6 months. The
obtained results are reported in Table 14.
TABLE-US-00015 TABLE 14 Yield a.sub.w (water a.sub.w (water CFU/g
.+-. SD CFU % activity) activity) Preparation T0 T3 T6 T6/T0 T0 T6
A-1 3.4 .+-. 0.2 .times. 10.sup.10 1.3 .+-. 0.2 .times. 10.sup.10
9.8 .+-. 5.7 .times. 10.sup.4 <10% A-2 3.5 .+-. 0.6 .times.
10.sup.10 6.4 .+-. 1.5 .times. 10.sup.9 <1.78 .times. 10.sup.2
<10% A-3 2.9 .+-. 0.7 .times. 10.sup.10 7.1 .+-. 2.5 .times.
10.sup.9 -- B-1 2.9 .+-. 0.7 .times. 10.sup.10 7.1 .+-. 2.5 .times.
10.sup.9 -- B-2 3.3 .+-. 0.3 .times. 10.sup.10 1.2 .+-. 0.1 .times.
10.sup.10 -- 0.153 0.276 B-3 6.3 .+-. 0.2 .times. 10.sup.10 1.9
.+-. 0.2 .times. 10.sup.10 1.8 .+-. 0.1 .times. 10.sup.10 27.7 .+-.
1.0 0.100 0.163 B-4 3.9 .+-. 0.2 .times. 10.sup.10 2.3 .+-. 0.6
.times. 10.sup.10 2.9 .+-. 0.7 .times. 10.sup.10 75.0 .+-. 19.2
0.120 0.313 B-5 4.6 .+-. 0.9 .times. 10.sup.10 4.9 .+-. 0.4 .times.
10.sup.10 -- 0.136 0.279 B-6 3.7 .+-. 0.5 .times. 10.sup.10 4.2
.+-. 0.3 .times. 10.sup.10 5.0 .+-. 0.6 .times. 10.sup.10 136.2
.+-. 14.8 0.143 0.182 B-7 2.9 .+-. 0.8 .times. 10.sup.10 1.9 .+-.
0.3 .times. 10.sup.10 2.6 .+-. 2.1 .times. 10.sup.10 91.1 .+-. 17.9
0.087 --
[0156] The table shows that the lyophilic products obtained in the
presence of trealose are stable at 4.degree. C. for 6 months and
that the water activity (a.sub.w) is always lower than 0.6.
Example 8
[0157] Compositions Comprising Lactobacillus paracasei LMG S-26420
in Sachets
[0158] The preparations in sachets comprise an amount of lyophilic
product, prepared as in Example 2 (preparations B), of 800 mg and
500 mg, corresponding respectively to about 3.times.10.sup.10 and
2.times.10.sup.10 living cells of Lactobacillus paracasei LMG
S-26420.
[0159] The lyophilic product was mixed with excipients,
preventively sieved, and the homogeneous mixture was divided in
sachets. The unitary compositions are reported in Table 15.
[0160] Among the described compositions, one comprises inuline,
another comprises xilo-oligosaccharide and the others a mixture of
fructo-oligosaccharides formed by a chain of fructose molecules
bound to a molecule of glucose (Actilight 950P).
TABLE-US-00016 TABLE 15 Comp. 1 Comp. 2 Comp. 3 Comp. 4 weight
weight weight weight Component (mg) (mg) (mg) (mg) Lactobacillus
paracasei 800 800 500 500 lyophilic LMG S-26420 Inulin 3200
Xilo-oligosaccharide 3200 506 506 Flavour (Passion fruit) 44 44
Silica 10 10 Astaxantine 160 Actilight 950P 2940 1720 Total weight
4000 4000 4000 4000
[0161] The compositions can comprise vitamins, like for example
vitamin E, vitamin B1, vitamin B2, vitamin B16.
[0162] The compositions in sachets can be suspended in water
solutions or in semi-solid foods.
Example 9
[0163] Compositions Comprising Lactobacillus paracasei LMG S-26420
in Tablets
[0164] The preparations in tablets containing an amount of 250 mg
of lyophilic product, corresponding to about 1.times.10.sup.10
living cells of Lactobacillus paracasei LMG S-26420, were obtained
by mixing a lyophilic with excipients preventively sieved on a net
of 800 micron. The mixture was then compressed in a Ronchi
compressing machine or an analogous machine by applying a
compressing force of about 13 KN.
[0165] The unitary composition of the tablets is reported in Table
16.
TABLE-US-00017 TABLE 16 Components Amount (mg) Lactobacillus
paracasei 250 (1 .times. 10.sup.10 .+-. CFU) LMG S-26420 Isomalto
278.0 Croscarmellose 9.9 Talc 5.5 Silica 3.8 Magnesium stearate 2.8
Total weight 550
* * * * *