U.S. patent application number 13/259894 was filed with the patent office on 2012-01-26 for bacterial strains having an outstanding ability to produce menaquinone.
This patent application is currently assigned to DANISCO A/S. Invention is credited to Patrick Boyaval, Pascal Fourcassie, Philippe Horvath.
Application Number | 20120020927 13/259894 |
Document ID | / |
Family ID | 42262660 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120020927 |
Kind Code |
A1 |
Fourcassie; Pascal ; et
al. |
January 26, 2012 |
Bacterial Strains Having an Outstanding Ability to Produce
Menaquinone
Abstract
The invention relates to bacterial strains having an outstanding
ability to produce menaquinone and to applications thereof.
Inventors: |
Fourcassie; Pascal;
(Poitiers, FR) ; Boyaval; Patrick; (Les Meziere,
FR) ; Horvath; Philippe;
(St-gervais-les-trois-clochers, FR) |
Assignee: |
DANISCO A/S
Copenhagen K
DK
|
Family ID: |
42262660 |
Appl. No.: |
13/259894 |
Filed: |
April 15, 2010 |
PCT Filed: |
April 15, 2010 |
PCT NO: |
PCT/EP2010/055002 |
371 Date: |
September 23, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61169494 |
Apr 15, 2009 |
|
|
|
Current U.S.
Class: |
424/93.4 ;
426/61; 435/133; 435/170; 435/252.1 |
Current CPC
Class: |
A23C 19/0323 20130101;
A61P 9/00 20180101; A23C 9/1234 20130101; A23Y 2240/21 20130101;
A23C 9/1236 20130101; C12P 7/66 20130101; A23L 33/135 20160801;
A23Y 2320/39 20130101; A61P 19/10 20180101; C12N 1/20 20130101;
C12R 1/46 20130101; A23V 2250/714 20130101; A61K 35/74 20130101;
A23V 2002/00 20130101; A61K 35/744 20130101; C12R 1/01 20130101;
A23V 2002/00 20130101; A23L 33/15 20160801; A23Y 2240/41 20130101;
A23V 2200/318 20130101; A23K 10/18 20160501; A61P 7/00 20180101;
A23V 2200/326 20130101; A61P 17/00 20180101 |
Class at
Publication: |
424/93.4 ;
435/133; 435/252.1; 435/170; 426/61 |
International
Class: |
A61K 35/74 20060101
A61K035/74; C12N 1/20 20060101 C12N001/20; C12P 1/04 20060101
C12P001/04; A23C 9/12 20060101 A23C009/12; A23C 9/123 20060101
A23C009/123; A23C 13/16 20060101 A23C013/16; A23C 19/08 20060101
A23C019/08; A23C 19/076 20060101 A23C019/076; A23C 9/20 20060101
A23C009/20; A23L 1/29 20060101 A23L001/29; A23C 11/10 20060101
A23C011/10; A61P 19/10 20060101 A61P019/10; A61P 9/00 20060101
A61P009/00; A61P 17/00 20060101 A61P017/00; A61P 7/00 20060101
A61P007/00; C12P 7/66 20060101 C12P007/66 |
Claims
1. Use of a strain selected from the group consisting of the strain
deposited under the Budapest Treaty on 24th of February 2009 at the
CNCM under number CNCM I-4128, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23476, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23477, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23478, or a variant thereof, and the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23479, or a variant thereof, for producing
menaquinone.
2. A strain selected from the group consisting of the strain
deposited under the Budapest Treaty on 24th of February 2009 at the
CNCM under number CNCM I-4128, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23476, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23477, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23478, or a variant thereof, and the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23479, or a variant thereof, for use in a
method for treatment of the human or animal body.
3. The strain or variant thereof according to claim 2, wherein said
method for treatment is a method for treatment of a disease
selected from the group comprising bone diseases such as
osteoporosis; vascular diseases such as cardiovascular diseases,
blood pressure dysfunctions and blood clotting; and skin diseases,
such as loss of skin elasticity.
4. Use of a strain selected from the group consisting of the strain
of deposited under the Budapest Treaty on 24th of February 2009 at
the CNCM under number CNCM I-4128, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23476, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23477, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23478, or a variant thereof, and the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23479, or a variant thereof, for preparing a
product selected from the group consisting of a dietary supplement,
a pharmaceutical preparation, a food preparation and a feed
preparation.
5. A product comprising a strain selected from the group consisting
of the strain deposited under the Budapest Treaty on 24th of
February 2009 at the CNCM under number CNCM I-4128, or a variant
thereof, the strain deposited under the Budapest Treaty on 24th of
March 2010 at the DSMZ under number DSM 23476, or a variant
thereof, the strain deposited under the Budapest Treaty on 24th of
March 2010 at the DSMZ under number DSM 23477, or a variant
thereof, the strain deposited under the Budapest Treaty on 24th of
March 2010 at the DSMZ under number DSM 23478, or a variant
thereof, and the strain deposited under the Budapest Treaty on 24th
of March 2010 at the DSMZ under number DSM 23479, or a variant
thereof.
6. The product according to claim 5, wherein said product is
selected from the group consisting of a dietary supplement, a
pharmaceutical preparation, a food preparation and a feed
preparation.
7. The product according to claim 5, wherein said product is a
dairy product selected from the group comprising a fermented milk,
a yogurt, a matured cream, a cheese, a fromage frais, a milk drink,
a dairy product retentate, a processed cheese, a cream dessert, a
cottage cheese or an infant milk.
8. The product according to claim 7, wherein said dairy product
comprises milk of animal and/or plant origin.
9. A method for enriching the menaquinone content of a product
comprising the step of adding to said product a strain selected
from the group consisting of the strain deposited under the
Budapest Treaty on 24th of February 2009 at the CNCM under number
CNCM I-4128, or a variant thereof, the strain deposited under the
Budapest Treaty on 24th of March 2010 at the DSMZ under number DSM
23476, or a variant thereof, the strain deposited under the
Budapest Treaty on 24th of March 2010 at the DSMZ under number DSM
23477 or a variant thereof, the strain deposited under the Budapest
Treaty on 24th of March 2010 at the DSMZ under number DSM 23478 or
a variant thereof, and the strain deposited under the Budapest
Treaty on 24th of March 2010 at the DSMZ under number DSM 23479 or
a variant thereof.
10. The method according to claim 9, wherein said product is
selected from the group consisting of a dietary supplement, a
pharmaceutical preparation, a food preparation and a feed
preparation.
11. A strain selected from the group consisting of the strain
deposited under the Budapest Treaty on 24th of February 2009 at the
CNCM under number CNCM I-4128, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23476, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23477, or a variant thereof, the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23478, or a variant thereof, and the strain
deposited under the Budapest Treaty on 24th of March 2010 at the
DSMZ under number DSM 23479, or a variant thereof, for use in a
method for improving calcium fixation on the bones, maintaining or
improving the bone structure and/or resistance, and/or improving
the bone development.
12. A strain of Lactococcus lactis subsp. cremoris deposited under
the Budapest Treaty on 24th of February 2009 at the CNCM under
number CNCM I-4128, or a variant thereof.
13. The strain or the variant thereof according to claim 12,
wherein said variant produces at least 7 .mu.g, particularly at
least 9 .mu.g, more particularly at least 11 .mu.g of menaquinone
per 100 g of milk fermented with said strain or variant thereof
when measured in a Test A.
14. The strain or the variant thereof according to claim 12,
wherein said variant produces at least 200 .mu.g, particularly at
least 230 .mu.g, more particularly at least 260 .mu.g of
menaquinone per 100 g of freeze-dried cells when measured in a Test
B.
15. The product according to claim 6, wherein said product is a
dairy product selected from the group comprising a fermented milk,
a yogurt, a matured cream, a cheese, a fromage frais, a milk drink,
a dairy product retentate, a processed cheese, a cream dessert, a
cottage cheese or an infant milk.
16. The product according to claim 15, wherein said dairy product
comprises milk of animal and/or plant origin.
17. The strain or the variant thereof according to claim 13 wherein
said variant produces at least 200 .mu.g, particularly at least 230
.mu.g, more particularly at least 260 .mu.g of menaquinone per 100
g of freeze-dried cells when measured in a Test B.
Description
FIELD OF THE INVENTION
[0001] The invention relates to bacterial strains having an
outstanding ability to produce menaquinone.
BACKGROUND OF THE INVENTION
[0002] Menaquinone or vitamin K2 is involved in the carboxylation
of certain glutamate residues in proteins to form
gamma-carboxyglutamate residues (abbreviated Gla-residues). The
modified residues are often (but not always) situated within
specific protein domains called Gla domains. Gla-residues are
usually involved in binding calcium. The Gla-residues are essential
for the biological activity of all known Gla-proteins (Furie B,
Bouchard B A, Furie B C; Blood 93 (6): 1798-808). To date, 14 human
proteins with Gla domains have been discovered, and they play key
roles in the regulation of three physiological processes, such as
blood coagulation (prothrombin (factor II), factors VII, IX, X,
protein C, protein S and protein Z); bone metabolism (osteocalcin,
also called bone Gla-protein (BGP), and matrix gla protein (MGP);
and vascular biology.
[0003] Accordingly, menaquinone deficiency may induce several
pathologies such as for example bleeding, coagulation dysfunctions,
osteoporosis . . . . Typically, the groups of patients which are
considered to be particularly exposed to a menaquinone deficiency
are the new born, the elderly, the patients having liver, bile or
intestinal dysfunctions, and patients having chronic antibiotic
treatment.
[0004] Several methods have been proposed in the prior art to
supplement the menaquinone deficiency of these patients. One of
these methods consists to administer to these patients
menaquinone-producing bacteria. Indeed, menaquinone is produced by
the bacteria of the intestinal flora, and in particular by the
bacteria of the species Escherichia coli, Bacillus subtilis and
Bacteroides subsp. Menaquinone is also produced by several lactic
acid bacteria, such as for example the bacteria of the genera
Bifidobacterium, Lactococcus, Leuconostoc, Enteroccocus and
Propionibacterium. In particular, a specific variant of Lactococcus
lactis subsp. cremoris having the ability to produce more
menaquinone than the corresponding wild type strain has been
recently disclosed in WO 2008/040793.
[0005] However, having strains capable of producing higher amounts
of menaquinone would be very interesting.
SUMMARY OF THE INVENTION
[0006] The invention relates to bacterial strains having an
outstanding ability to produce menaquinone. Indeed, during their
research, the inventors have found that the particular strains
according to the invention present an exceptional ability to
produce menaquinone compared to the other strains known to date.
These strains have been deposited under the Budapest Treaty under
the following accession numbers: CNCM I-4128, DSM 23476, DSM 23477,
DSM 23478, DSM 23479.
[0007] The invention particularly relates to the use of these
strains and variants thereof for producing menaquinone.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The invention relates to bacterial strains having an
outstanding ability to produce menaquinone as compared to other
strains known to date.
[0009] An object of the invention concerns a strain of Lactococcus
lactis subsp. cremoris deposited by Danisco France SAS (20, rue de
Brunel, 75017 Paris, France) under the Budapest Treaty on 24th of
February 2009 at the Collection Nationale de Cultures de
Microorganismes (CNCM, Institut Pasteur, 25, rue du Docteur Roux,
75724 Paris cedex 15, France) under number CNCM I-4128, or a
variant thereof. The strain CNCM I-4128 or variants thereof produce
high quantities of menaquinone. In particular, the inventors have
shown that the strain CNCM I-4128 produce at least 7 .mu.g of
menaquinone per 100 g of milk fermented with said strains when
measured in a Test A.
[0010] Typically, the variants of the strain CNCM I-4128 produce at
least 7 .mu.g, particularly at least 8 .mu.g, still particularly at
least 9 .mu.g, more particularly at least 10 .mu.g, still more
particularly at least 11 .mu.g and most particularly at least 12
.mu.g of menaquinone per 100 g of milk fermented with said variant
when measured in a Test A according to the invention.
[0011] Test A is fully described in the experimental section.
Briefly, Test A comprises the following steps: [0012] inoculating
two flasks comprising 100 mL of skimmed UHT milk, with 5.10.sup.4
to 10.sup.7 cfu/mL of the strain to be tested, [0013] incubating
the inoculated flasks without stirring at a constant temperature
selected in the range from 23.degree. C. to 30.degree. C. in a
water bath, [0014] measuring and recording the evolution of the pH
in one of the two flasks with a pH probe of a Cinac System
(Ysebaert system), [0015] stopping the incubation when the pH
reaches 4.60+/-0.1 by cooling down to 6.degree. C. the flask
wherein the pH has not been measured, [0016] storing said flask at
6.degree. C. during 14 h to 20 h, [0017] homogenizing the milk
manually, [0018] performing a chemical extraction of menaquinone
and measuring the quantity of menaquinone following the protocol
P.
[0019] In another embodiment of the invention, the variants of the
strain CNCM I-4128 produce at least 200 .mu.g, particularly at
least 230 .mu.g, more particularly at least 260 .mu.g of
menaquinone per 100 g of freeze-dried cells when measured in a Test
B. Test B is fully described in the experimental section. Briefly,
Test B comprises the following steps: [0020] culturing 1.10.sup.2
to 1.10.sup.7 cfu/ml of the strain to be tested during 14 h to 20 h
in 50 ml of M17-lactose broth medium (Biokar BK088HA) at 30.degree.
C., [0021] centrifuging 25 mL of the culture at 8000 rpm during 10
minutes, [0022] discarding the supernatant and resuspending the
pellet with 25 mL of a tryptone salt solution (0.1% tryptone, 0.85%
salt), [0023] centrifuging the resuspended culture at 8000 rpm
during 10 minutes, [0024] discarding the supernatant and
resuspending the pellet in 25 ml of reconstituted milk powder at
10% (w/w), [0025] placing the cell suspension in a freeze dryer,
[0026] obtaining between 2 and 4 grams of freeze-dried cells,
[0027] performing the chemical extraction of menaquinone and
measuring the quantity of menaquinone following the protocol P.
[0028] Protocol P is the following:
[0029] A sample consisting of 10 ml of fermented milk (for test A)
or 10 mL of a ten times dilution in ethanol/water (50/50 V/V) of
freeze-dried cells (for test B) is mixed with 5 ml of HCl 1N. The
sample is then heated at 100.degree. C. during 10 min in a water
bath. Then, 10 ml of isopranol are added in the tube. The tube is
placed 10 min in a water bath at 22.degree. C. (+/-3.degree. C.)
equipped with ultra sounds. 5 ml of hexane are added in the tube.
The tube is mixed by vortexing during 5 min.
[0030] The suspension is centrifuged at 4600 rpm during 5 min. Then
the organic phase is harvested and again centrifuged during 5 min
at 4600 rpm. The organic phase is then harvested and concentrated
with a speed vac system in order to obtain a dry product. The dry
product is rehydrated with 1 ml ethanol and filtrate on 0.45 .mu.m
filter. This extract is injected in a HPLC system. Separation and
detection of menaquinone are performed using methods described in
Hojo K. et al., "Quantitative measurement of
tetrahydromenaquinone-9 cheese fermented by propionibacteria", J.
dairy Science, 2007, 90, 9. 4078-4083. The detection is performed
by a fluorometer after post-column reduction of menaquinone by Zn.
Vitamin K1 is used as internal standard for extraction/purification
steps and MK-4 (Sigma V9378) is used as external calibration for
quantification.
[0031] The invention also concerns a strain of Lactococcus lactis
spp. cremoris deposited by Danisco Deutschland GmbH
(Busch-Johannsen-Str. 1, 25899 Niebull, Germany) under the Budapest
Treaty on 24th of March 2010 at the Deutsche Sammlung von
Mikroorganismen and Zellkulturen (DSMZ, Inhoffenstr. 7B, D-38124
Braunschweig, Germany) under number DSM 23476, or a variant
thereof.
[0032] The strain DSM 23476 or variants thereof produce high
quantities of menaquinone. The variants of the strain DSM 23476
typically produce at least 25 .mu.g of menaquinone per g of
freeze-dried cells when measured in a Test C according to the
invention.
[0033] In one embodiment, the variants of the strain DSM 23476
typically produce at least 30 .mu.g, particularly at least 35
.mu.g, still particularly at least 40 .mu.g, more particularly at
least 45 .mu.g, still more particularly at least 50 .mu.g, again
more particularly at least 55 .mu.g, and most particularly at least
60 .mu.g of menaquinone per g of freeze-dried cells when measured
in a Test C according to the invention.
[0034] The invention also concerns a strain of Lactococcus lactis
spp lactis deposited by Danisco Deutschland GmbH
(Busch-Johannsen-Str. 1, 25899 Niebull, Germany) under the Budapest
Treaty on 24th of March 2010 at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ, Inhoffenstr. 7B, D-38124
Braunschweig, Germany) under number DSM 23477, or a variant
thereof.
[0035] The strain DSM 23477 or variants thereof produce high
quantities of menaquinone.
[0036] The variants of the strain DSM 23477 typically produce at
least 15 .mu.g of menaquinone per g of freeze-dried cells when
measured in a Test C according to the invention.
[0037] In one embodiment, the variants of the strain DSM 23477
typically produce at least 20 .mu.g, particularly at least 25
.mu.g, still particularly at least 30 .mu.g, more particularly at
least 35 .mu.g, still more particularly at least 40 .mu.g of
menaquinone per g of freeze-dried cells when measured in a Test C
according to the invention.
[0038] The invention also concerns a strain of Lactococcus lactis
spp cremoris deposited by Danisco Deutschland GmbH
(Busch-Johannsen-Str. 1, 25899 Niebull, Germany) under the Budapest
Treaty on 24th of March 2010 at the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ, Inhoffenstr. 7B, D-38124
Braunschweig, Germany) under number DSM 23478, or a variant
thereof.
[0039] The strain DSM 23478 or variants thereof produce high
quantities of menaquinone.
[0040] The variants of the strain DSM 23478 typically produce at
least 30 .mu.g of menaquinone per g of freeze-dried cells when
measured in a Test C according to the invention.
[0041] In one embodiment, the variants of the strain DSM 23478
typically produce at least 35 .mu.g, particularly at least 40
.mu.g, still particularly at least 45 .mu.g, more particularly at
least 50 .mu.g, still more particularly at least 55 .mu.g of
menaquinone per g of freeze-dried cells when measured in a Test C
according to the invention.
[0042] Briefly, Test C according to the invention comprises the
following steps: 1) Culturing the strain: 1.A) the strain stored in
a viable physiological state at temperature below -20.degree. C. is
first sub-cultured in a synthetic medium (Lactose 50 g/l, Yeast
Extract powder 36 g/l, Mn SO.sub.4H.sub.20 1 g/l, Mg SO.sub.47
H.sub.20 1 g/l) at 30.degree. C.+/-2.degree. C. overnight,
[0043] 1.B) from 2% to 5% (V/V) of the first culture obtained after
step 1.A) is then sub-cultured in synthetic medium from 10 h to 24
h, at a temperature comprised between 25.degree. C. and 35.degree.
C., a pH maintained in the range 5.0-7.5 by alkali addition, and
dissolved oxygen kept below 2% during the entire step,
[0044] 2) Concentrating the strain: the cultured strain obtained
after step 1.B) is concentrated with a centrifuge separator until a
concentration comprised between 2.10.sup.10 cfu/ml and 6.10.sup.11
cfu/ml is obtained; the concentrate thus obtained is then cooled at
a temperature below 12.degree. C.;
[0045] 3) Preserving the strain: 30 to 70% (w/w) of skimmed milk
powder is directly added to the concentrate, frozen at -55.degree.
C., pelletized to obtain frozen pellets, and freeze dried in a
freeze drier to achieve a Aw with in the range 0.05-0.35 (measured
with 3TE Aqualab Ltd device),
[0046] 4) Extracting and measuring the quantity of menaquinone
following the protocol Q according to the invention.
[0047] Protocol Q comprises the following steps: [0048] 1)
Extracting menaquinone (all the steps of the extraction are carried
out in subdued light to prevent the deterioration of K vitamins):
[0049] Put 10 mL of a solution containing 0.5 g of freeze dried
material in 100 mL of nanopure water in a 50 mL amber Falcon tube
(O30.times.115 mm--Ref 525-0397 VWR), [0050] Add 1004 of internal
standard (Vitamin K1 at 20 .mu.g/mL in ethyl alcohol) and 5 mL of
hydrochloric acid 1N, [0051] Put the tube in a 100.degree. C. bath
during 10 min, [0052] After cooling, add 10 mL of isopropanol,
[0053] Sonicate the sample during 10 min, [0054] Add 5 mL of
hexane, shake the sample for 5 min, Centrifuge 5 min at 3760 g and
collect the organic layer (upper layer) in a 15 mL amber Falcon
tube (O17.times.120 mm--Ref 525-0395 VWR), Repeat this step twice,
[0055] After collecting the two organic layers, they are evaporated
to dryness in a vacuum evaporator Speed-Vac (about 30 min), [0056]
Dissolve the residue in 1 mL of ethanol, [0057] The sample is
filtered through a Nylon 0.45 .mu.m filter and placed in a HPLC
vial, [0058] 2) measuring the quantity of menaquinone in the sample
by HPLC.
[0059] HPLC Analysis:
[0060] High performance liquid chromatography coupled to a
fluorimetry detector is used in order to analyse vitamin K.
Different forms of vitamin K are separated on a reverse phase
column and reduced through a post column.
[0061] Identified and measured forms of vitamin K2:
TABLE-US-00001 K.sub.2 vitamin (MK-n) MK-4 MK-5 MK-6 MK-7 MK-8 MK-9
MK-10 ##STR00001## K.sub.1 vitamin used as internal standard
##STR00002## ##STR00003##
[0062] Products and Solutions: [0063] K1 Vitamin, Sigma V3501-1G,
CAS 84-80-0 [0064] K2 Vitamin, (MK-4), Sigma V9378-250MG, CAS
11032-49-8
[0065] Mobile Phase: [0066] In a flask with 830 mL of methanol and
170 mL of ethanol, add 0.68 g of sodium acetate, 1.36 g of zinc
chloride and 300 .mu.L of acetic acid. [0067] Shake the mix and
sonicate it during 10 min.
[0068] The analysis is carried out by an Agilent HPLC 1100 in
reverse phase with a fluorescence detector. In order to improve
vitamin K's sensibility detection, it's reduced after separation in
hydroquinone by chemical reduction with zinc metal. Hydroquinones
are more fluorescent than quinones.
TABLE-US-00002 Reduction: Quinone + 2 e.sup.- + 2 H.sup.+
.fwdarw.H.sub.2Q E .degree. = +0.70 V (Hydroquinone) Oxidation: Zn
.fwdarw.Zn.sup.2+ + 2e.sup.- E .degree. = -0.76 V Balance: Quinone
+ 2 H.sup.+ + Zn.fwdarw.H.sub.2Q (Hydroquinone) + Zn.sup.2+
[0069] Analytical Conditions: [0070] Column: Capcell Pak 5 .mu.m
SG-C18, 250.times.4.6 mm (Phenomenex) [0071] Post column reduction:
Reactor post column zinc, 20.times.4 mm. [0072] Mobile phase: 83%
of methanol [0073] 17% of ethanol [0074] 5 mM of sodium acetate
[0075] 10 mM of ZnCl.sub.2 [0076] 5 mM of acetic acid [0077] Flow:
1 mL/min (isocratic elution) [0078] Column temperature: 55.degree.
C. [0079] Detector: .lamda..sub.ex=220 nm, .lamda..sub.em=436 nm
[0080] Injection volume: 10 .mu.L
[0081] Menaquinone Quantification:
[0082] The internal standard, Vit K1, is used to calculated the
yield of extraction/purification for each sample preparation. For
calibration of the technique, a calibration curve has been obtained
for K2 Vitamin, (MK-4), Sigma V9378-250MG. The response coefficient
of MK-4 is applied for the other form of MK. It allows to transform
peak surface area into Mk-4 equivalent concentration in the sample
for each MK-form. Furthermore, for each form of MK's, the
concentration in mass for each form is calculated taking into
account the differences in molecular mass between menaquinone MK-4
and MK form considered. For example, the molecular mass of MK4 is
512 g/mol, the molecular mass of MK9 is 852 g/mol, therefore 1
.mu.g of MK4 will give exactly the same peak surface area than 1.66
.mu.g of MK9. This principle of calculation is used for each MK
form.
[0083] The invention also concerns a strain of Propionibacterium
freudenreichii subsp. Shermanii deposited by Danisco Deutschland
GmbH (Busch-Johannsen-Str. 1, 25899 Niebull, Germany) under the
Budapest Treaty on 24th of March 2010 at the Deutsche Sammlung von
Mikroorganismen and Zellkulturen (DSMZ, Inhoffenstr. 7B, D-38124
Braunschweig, Germany) under number DSM 23479, or a variant
thereof.
[0084] The strain DSM 23479 or variants thereof produce high
quantities of menaquinone. A variant of the strain DSM 23479
typically produces at least 15 .mu.g of menaquinone per 100 g of
milk fermented with said variant when measured in a Test D
according to the invention.
[0085] In one embodiment, a variant of the strain DSM 23479
typically produces at least 15 .mu.g, particularly at least 20
.mu.g, still particularly at least 25 .mu.g of menaquinone per 100
g of milk fermented with said variant when measured in a Test D
according to the invention.
[0086] Test D according to the invention comprises the following
steps: [0087] inoculating two flasks comprising 100 mL of a skimmed
UHT milk supplemented with 1.6 ml of pure D-L lactate and 0.2% of
yeast extract powder and heat treated 20 minutes at 110.degree. C.,
with 5.10.sup.4 to 10.sup.7 cfu/mL of the strain to be tested,
[0088] incubating the inoculated flasks without stirring at a
constant temperature selected in the range from 23.degree. C. to
30.degree. C. in a water bath, [0089] measuring and recording the
evolution of the pH in one of the two flasks with a pH probe of a
Cinac System (Ysebaert system), [0090] stopping the incubation when
the pH reaches 4.60+/-0.1 by cooling down to 6.degree. C. the flask
wherein the pH has not been measured, [0091] storing said flask at
6.degree. C. during 14 h to 20 h, [0092] homogenizing the milk
manually, [0093] performing a chemical extraction of menaquinone
and measuring the quantity of menaquinone following the protocol P
according to the invention.
[0094] According to the invention, by "variant" it is meant: [0095]
a natural variant of the strain according to the invention, i.e. a
variant obtained spontaneously from the strain according to the
invention after incubation in a selection medium. A natural variant
is thus obtained without any genetic manipulation of the operator
but only by natural mutation of the strain and selection of the
strain in an appropriate medium, or [0096] a variant of the strain
according to the invention comprising at least one mutation in
their genome, said mutation being induced by genetic engineering,
for instance by directed mutagenesis or random mutagenesis. For
instance, random mutagenesis can be performed with UV radiations or
mutagenic compounds such as nitrous acid, ethyl-methanesulfonate,
N-Methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea,
acridine orange, proflavine.
[0097] By "mutation" according to the invention, it is meant the
addition, deletion, or the substitution of at least one nucleotide
in the genome of the strain according to the invention.
[0098] The strains according to the invention or variants thereof,
are thus very interesting in term of health benefits, but also in
term of marketing. For example, in Europe when a product contains
at least 11.5 .mu.g of menaquinone per serving size, more
particularly per 100 g or per 100 mL of product, it can be labelled
as "source of" vitamin K2.
[0099] Another object of the invention concerns the use of the
strains or variants thereof according to the invention for
producing menaquinone. An application of the invention is the use
of the outstanding properties of the strains and variants thereof
according to the invention to produce important amounts of
menaquinone at an industrial scale. The menaquinone can then be
extracted from the cultures of strains according to the invention
and be used in all kinds of preparations, for instance in
pharmaceutical preparations, feed and food preparations such as a
dairy product, or dietary supplements.
[0100] The invention thus also concerns a method for producing
menaquinone, comprising the step of culturing a strain or a variant
thereof according the invention in a substrate. Said substrate can
be selected from any appropriate substrate known by the skilled
person. Examples of appropriate substrates are milk substrates, in
particular selected from the group consisting of natural or
reconstituted milk, skimmed or otherwise, milk-based media and
media based on products of dairy origin.
[0101] Another object of the invention is a strain or a variant
thereof according to the invention, for use in a method for
treatment of the human or animal body.
[0102] Typically, the strains or variants thereof according to the
invention are useful in the treatment of the diseases associated
with a deficiency in menaquinone, as for instance bone, vascular
and/or skin health diseases such as osteoporosis, cardiovascular
diseases, blood pressure dysfunctions, blood clotting, loss of skin
elasticity.
[0103] The invention thus relates to a method for treating a
disease selected from the group comprising bone diseases such as
osteoporosis; vascular diseases such as cardiovascular diseases,
blood pressure dysfunctions and blood clotting; and skin diseases,
such as loss of skin elasticity; said method comprising the step of
administering to a patient in need thereof, a therapeutically
effective amount of a strain or of a variant thereof according to
the invention.
[0104] The invention also relates to a method for improving calcium
fixation on the bones, maintaining or improving the bone structure
and/or resistance, and/or improving the bone development, said
method comprising the step of administering to a patient in need
thereof, an effective amount of a strain or of a variant thereof
according to the invention. Alternatively, the invention relates to
the strains or variants thereof according to the invention, for use
in a method for improving calcium fixation on the bones,
maintaining or improving the bone structure and/or resistance,
and/or improving the bone development.
[0105] The term "treating" or "treatment", as used herein, means
reversing, alleviating, inhibiting the progress of, or preventing
the disorder or condition to which such term applies.
[0106] As used herein, "patient" refers to a human or animal that
may benefit from the administration of a strain or a variant
thereof as recited herein.
[0107] By a "therapeutically effective amount" of a strain or a
variant thereof as described previously, it is meant a sufficient
amount to treat the disease, at a reasonable benefit/risk ratio
applicable to any medical treatment.
[0108] In a particular embodiment of the method for treatment of
the invention, the strain or a variant thereof can be used alive or
under a specific preserved state. When the strain or variant
thereof is used in a preserved state, the strain has been
preferably previously cultured to enrich its content in
menaquinone. By "preserved state" it is meant a strain that has
been dried, freeze-dried or frozen for example. The strain or a
variant thereof may also be used after having been inactivated, for
instance by heat treatment, by chemical treatment and/or by other
treatments known by the skilled person in the art.
[0109] Another aspect of the invention concerns the preparation of
products enriched in menaquinone by using the strains or variants
thereof according to the invention. These products are thus
particularly useful for supplementing any deficiency in menaquinone
occurring in a patient in need thereof. Such deficiency in
menaquinone is for instance encountered in the newborn infants,
individuals who suffer from liver damage or disease (i.e.
alcoholics), people with cystic fibrosis, inflammatory bowel
diseases or those who have recently had abdominal surgeries. Groups
which may suffer from secondary vitamin K deficiency include
bulimics, those on stringent diets and those taking
anticoagulants.
[0110] Accordingly, the invention concerns the use of a strain or a
variant thereof according to the invention for preparing a product.
This product is typically selected from the group consisting of a
dietary supplement, a pharmaceutical preparation, a food
preparation and a feed preparation. In a particular embodiment, in
these products, the strain or a variant thereof can be used alive
or under a specific preserved state. When the strain or variant
thereof is used in a preserved state, the strain has been
preferably previously cultured to enrich its content in
menaquinone. By "preserved state" it is meant a strain that has
been dried, freeze-dried or frozen for example. The strain or a
variant thereof may also be used after having been inactivated, for
instance by heat treatment, by chemical treatment and/or by other
treatments known by the skilled person in the art.
[0111] The invention also relates to a product typically selected
from the group comprising of a dietary supplement, a pharmaceutical
preparation, a food preparation and a feed preparation, wherein
said product comprises a strain or a variant thereof according to
the invention.
[0112] According to the invention, by "dietary supplement" it is
meant a product or a composition that is intended to supplement the
diet of the human or of an animal. A dietary supplement according
to the invention is typically intended for ingestion in pill,
capsule, tablet, or liquid form.
[0113] According to the invention, by "pharmaceutical preparation"
it is meant a preparation that is intended to be used in a method
for treatment of the human or animal body. In the context of the
invention, the term "treatment", as used herein, means reversing,
alleviating, inhibiting the progress of, or preventing a disorder
or condition. Typically, the pharmaceutical preparation according
to the invention comprises the strain or a variant thereof
according to the invention together with a pharmaceutically
acceptable carrier.
[0114] According to the invention, by "food preparation" it is
meant a preparation that is intended to feed a human.
[0115] According to the invention, by "feed preparation" it is
meant a preparation that is intended to feed an animal.
[0116] In a particular embodiment of the invention, said product is
a food preparation. More particularly, the food preparation is a
dairy product. Within the meaning of the invention, by "dairy
product" it is meant fermented milk, a yogurt, a matured cream, a
cheese, a fromage frais, a milk drink, a dairy product retentate, a
processed cheese, a cream dessert, a cottage cheese or an infant
milk. Still typically, the dairy product according to the invention
comprises milk of animal and/or plant origin.
[0117] Another aspect of the invention concerns the application of
the strains and variants thereof according to the invention to
enrich the content of a product in menaquinone. Accordingly, the
invention relates to a method for enriching the menaquinone content
of a product selected from the group consisting of a dietary
supplement, a pharmaceutical preparation, a food preparation and a
feed preparation, comprising the step of adding to said product a
strain or a variant thereof according to the invention. In a
particular embodiment of this method, the strain or a variant
thereof can be used alive or under a specific preserved state. When
the strain or variant thereof according to the invention is used in
a preserved state, the strain has been preferably previously
cultured to enrich its content in menaquinone. By "preserved state"
it is meant a strain that has been dried, freeze-dried or frozen
for example. The strain or a variant thereof may also be used after
having been inactivated, for instance by heat treatment, by
chemical treatment and/or by other treatments known by the skilled
person in the art.
[0118] The present invention is better illustrated below using the
examples which follow. These examples are given only by way of
illustration of the subject-matter of the invention, of which they
in no way constitute a limitation.
EXAMPLES
Example 1
Measurement of the Production of Menaquinone by the Strain CNCM
I-4128, Following TEST B
[0119] The strain CNCM I-4128 has been compared to 23 other strains
belonging to the Lactococcus lactis species for their production of
menaquinone (following TEST B).
[0120] For each strain, the following protocol has been followed:
[0121] culturing 1.10.sup.2 to 1.10.sup.7 cfu/ml of the strain
during 18 h in 50 ml of M17-lactose broth medium (Biokar BK088HA)
at 30.degree. C., [0122] centrifuging 25 mL of the culture at 8000
rpm during 10 minutes, [0123] discarding the supernatant and
resuspending the pellet with 25 mL of a tryptone salt solution
(0.1% tryptone, 0.85% salt), [0124] centrifuging the resuspended
culture at 8000 rpm during 10 minutes, [0125] discarding the
supernatant and resuspending the pellet in 25 ml of reconstituted
milk powder at 10% (w/w), [0126] placing the cell suspension in a
freeze dryer, [0127] obtaining between 2 and 4 grams of
freeze-dried cells.
[0128] The chemical extraction of menaquinone and the measurement
of its quantity have been performed according to the following
protocol:
[0129] The freeze-dried cells obtained in Test B are diluted ten
times in ethanol/water (50/50 V/V). 10 mL of this dilution are
mixed with 5 ml of HCl 1N. The sample is heated at 100.degree. C.
during 10 min in a water bath. Then, 10 ml of isopranol are added
in the tube. The tube is placed 10 min in a water bath at
22.degree. C. (+/-3.degree. C.) equipped with ultra sound. 5 ml of
hexane are added in the tube. The tube is mixed by vortexing during
5 min.
[0130] The suspension is centrifuged at 4600 rpm during 5 min. Then
the organic phase is harvested and again centrifuged during 5 min
at 4600 rpm. The organic phase is then harvested and concentrated
with a speed vac system in order to obtain a dry product. The dry
product is rehydrated with 1 ml ethanol and filtrate on 0.45 .mu.m
filter. This extract is injected in a HPLC system. Separation and
detection of menaquinone are performed using methods described in
Hojo K. et al., "Quantitative measurement of
tetrahydromenaquinone-9 cheese fermented by propionibacteria", J.
dairy Science, 2007, 90, 9. 4078-4083. The detection is performed
by a fluorometer after post-column reduction of menaquinone by Zn.
Vitamin K1 is used as internal standard for extraction/purification
steps and MK-4 (Sigma V9378) is used as external calibration for
quantification.
[0131] The results are presented in Table 1, which shows the
difference of production of menaquinone between the CNCM I-4128 and
23 other strains belonging to the Lactococcus lactis species (which
were Lactococcus lactis subsp. cremoris or Lactococcus lactis
subsp. lactis strains). Data are expressed in .mu.grams per 100
grams of freeze dried cells.
TABLE-US-00003 TABLE 1 Comparison of production of menaquinone
(Vit. K2) for 24 strains of Lactococcus lactis Total vitamin K2
expressed in Strains .mu.g/100 g of freeze dried cells 1 78 2 49
CNCM I-4128 267 3 91 4 48 5 60 6 59 7 79 8 62 9 94 10 42 11 68 12
38 13 12 14 57 15 73 16 68 17 79 18 28 19 36 20 130 21 73 22 63 23
94
[0132] The results of this experiment clearly show that the strain
CNCM I-4128 has an outstanding ability to produce menaquinone
compared to the 23 other strains tested.
Example 2
Measurement of the Production of Menaquinone by the Strain CNCM
I-4128, Following TEST A
[0133] The measure of the production of menaquinone by the strain
CNCM I-4128 in the milk (following TEST A) has been repeated 11
times, according to the following protocol: [0134] inoculating two
flasks comprising 100 mL of skimmed UHT milk, with 5.10.sup.4 to
10.sup.7 cfu/mL of the strain CNCM I-4128, [0135] incubating the
inoculated flasks without stirring at a constant temperature
selected in the range from 23.degree. C. to 30.degree. C. in a
water bath, [0136] measuring and recording the evolution of the pH
in one of the two flasks with a pH probe of a Cinac System
(Ysebaert system), [0137] stopping the incubation when the pH
reaches 4.60+/-0.1 by cooling down to 6.degree. C. the flask
wherein the pH has not been measured, [0138] storing said flask at
6.degree. C. during 14 h to 20 h, [0139] homogenizing the milk
manually.
[0140] The chemical extraction of menaquinone and the measurement
of its quantity have then been performed according to the following
protocol: 10 ml of milk obtained previously have been mixed with 5
ml of HCl 1N. The same protocol as in Example 1 has then been
followed to perform the extraction of menaquinone and to measure
its content.
[0141] Results are presented in table 2 hereinafter:
TABLE-US-00004 TABLE 2 Production of vitamin K2 by CNCM I-4128
during milk maturation for temperature range between 23.degree. C.
and 30.degree. C., results for 11 independent experiments Total
vitamin K2 expressed in Experiment n.degree. .mu.g/100 g of
fermented milk 1 7.3 2 9.9 3 12 4 12.3 5 15.2 6 9.8 7 12.2 8 12 9
16.3 10 8.7 11 11.6
[0142] These results show that the average production of CNCM
I-4128 is 11.6 .mu.g/100 g of fermented milk with a standard
deviation of 2.5 .mu.g/100 g.
Example 3
Measurement of the Production of Menaquinone by Strains CNCM
I-4128, DSM 23476, DSM 23477 and DSM 23478 by test C
[0143] CNCM I-4128:
[0144] Freeze dried material has been produced according to test C
and the menaquinone content in the freeze dried product has been
evaluated thanks to protocol Q for extraction/purification and
dosage.
TABLE-US-00005 TABLE A vitamin K2 concentration for CNCM-I-4128 -
test C and protocol Q coef- fi- Average cient MK- MK- MK- Total K2
of 6 7 MK-8 MK-9 10 K2 .mu.g/g of vari- Sample ng/g ng/g ng/g ng/g
ng/g .mu.g/g LYO ation CNCM 1865 7443 48307 155025 4812 374 378.1
3.6% I-4128 1736 6593 43810 156743 4061 367 2139 6912 57037 158733
4171 393
[0145] DSM 23476 and DSM 23477:
[0146] Freeze dried material has been produced according to test C
and the menaquinone content in the freeze dried product has been
evaluated thanks to protocol Q for extraction/purification and
dosage.
TABLE-US-00006 TABLE B vitamin K2 concentration for DSM 23476 and
DSM 23477 - test C and protocol Q - two trials test C+ protocol Q
per strain Average MK- MK- Total K2 coefficient 6 7 MK-8 MK-9 K2
.mu.g/g de of Sample ng/g ng/g ng/g ng/g .mu.g/g LYO variation DSM
4026 5451 16842 24928 51.25 45.2 13.2% 23476 3623 4965 14361 16372
39.32 Trial n.sup.o1 3698 5008 15866 20416 44.99 DSM 5104 7199
22166 30561 65.03 62.3 8.3% 23476 4860 6565 19144 25782 56.35 Trial
n.sup.o2 5649 8027 23437 28541 65.66 DSM 1960 3980 13145 10208
29.30 28.4 4.0% 23477 1795 3903 11518 9898 27.12 Trial n.sup.o1
1690 3855 12719 10529 28.80 DSM 1422 3309 15970 18408 39.11 42.1
11.1% 23477 1308 3176 16598 18554 39.64 Trial n.sup.o2 1290 3093
19051 23975 47.41
[0147] DSM 23478:
[0148] Freeze dried material has been produced according to test C
and the menaquinone content in the freeze dried product has been
evaluated thanks to protocol Q for extraction/purification and
dosage.
TABLE-US-00007 TABLE C vitamin K2 concentration for DSM 23478 - for
different extractions/purification step with the same freeze dried
material obtained with Test C. MK- MK-4 MK-5 MK-6 MK-7 MK-8 MK-9 10
Total K2 coefficient of Test ng/g ng/g ng/g ng/g ng/g ng/g ng/g
.mu.g/g variation 1 135.8 <100 178.7 1827.2 17597.1 60772.4
792.4 81.3 79.3 2.9% 2 124.1 <100 205.6 1935.2 18461.4 59126.8
695.7 80.5 3 204.2 <100 <100 1806.3 15680.9 57854.0 555.1
76.1 4 176.0 <100 450.4 1941.5 16657.6 59727.2 618.8 79.6
Example 4
Measurement of the Production of Menaquinone by DSM 23479 by Test
D
[0149] Strain DSM 23479 has been cultured as described in test D.
Menaquinone production has then be measured according to protocol
P. Results are presented in the table D hereinafter:
TABLE-US-00008 TABLE D Production of Vitamin K2 by DSM 23479 - test
D MK- MK- MK- MK- MK- K2 totale 4 5 6 7 8 MK- .mu.g/ Test ng/ ng/
ng/ ng/ ng/ MK-9 10 100 mL # mL mL mL mL mL ng/mL ng/mL (.mu.g/100
g) 1 <1 <1 <1 <1 <1 3.8 261.4 26.5
* * * * *