Canadian Patents Database / Patent 2326405 Summary

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(12) Patent: (11) CA 2326405
(54) English Title: NOVEL GENETICALLY MODIFIED LACTIC ACID BACTERIA HAVING MODIFIED DIACETYL REDUCTASE ACTIVITIES
(54) French Title: NOUVELLES BACTERIES D'ACIDE LACTIQUE, MODIFIEES GENETIQUEMENT ET POSSEDANT DES ACTIVITES DE DIACETYLE REDUCTASE MODIFIEES
(51) International Patent Classification (IPC):
  • C12N 15/01 (2006.01)
  • C12N 1/20 (2006.01)
  • C12N 9/04 (2006.01)
  • C12N 15/74 (2006.01)
(72) Inventors :
  • HENRIKSEN, CLAUS MAXEL (Denmark)
  • NILSSON, DAN (Denmark)
  • WALFRIDSSON, MATS (Sweden)
(73) Owners :
  • CHR. HANSEN A/S (Denmark)
(71) Applicants :
  • CHR. HANSEN A/S (Denmark)
(74) Agent: BLAKE, CASSELS & GRAYDON LLP
(74) Associate agent: BLAKE, CASSELS & GRAYDON LLP
(45) Issued: 2011-01-04
(86) PCT Filing Date: 1999-04-20
(87) Open to Public Inspection: 1999-10-28
Examination requested: 2001-03-07
(30) Availability of licence: N/A
(30) Language of filing: English

(30) Application Priority Data:
Application No. Country/Territory Date
0552/98 Denmark 1998-04-21
60/082,566 United States of America 1998-04-21
199801697 Denmark 1998-12-21

English Abstract



Genetically modified lactic acid
bacteria having a reduced or lacking
or enhanced diacetyl reductase activity,
acetoin reductase activity and/or
butanediol dehydrogenase activity are
provided. Such bacteria are used in
starter cultures in the production of food
products including dairy products where
it is desired to have a high content of
diacetyl and for reducing or completely
removing diacetyl in beverages including
beers, fruit juices and certain types of
wine, where the presence of diacetyl is
undesired.


French Abstract

Ces bactéries d'acide lactique, modifiées génétiquement, possèdent une activité de diacétyle réductase, d'acétoïne réductase et/ou de butanediol déshydrogénase, réduite ou accrue, ou sont dépourvues de cette activité. On utilise de telles bactéries dans des cultures de départ dans la production de produits alimentaires, notamment des produits laitiers dans lesquels on souhaite obtenir une forte teneur en diacétyle ; on utilise également ces bactéries pour réduire ou enlever complètement le diacétyle dans des boissons, notamment la bière, les jus de fruits et certains types de vins, lorsque la présence de diacétyle n'est pas souhaitée.


Note: Claims are shown in the official language in which they were submitted.


37
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A genetically modified lactic acid bacterium of a Leuconostoc species that,
relative to the
lactic acid bacterium from which it is derived; is modified to have a
reduction of at least 25% of
diacetyl reductase activity, acetoin reductase activity and butanediol
dehydrogenase activity in
the presence of NADH, NADPH, NAD+ or NADP+, wherein the genetically modified
bacterium
is generated by a process comprising the steps of:
(i) subjecting the lactic acid bacterium to genetic modification by a
mutagenization
treatment or recombinant DNA technology;
(ii) measuring the genetically modified bacterium for diacetyl reductase
activity,
acetoin reductase activity and butanediol dehydrogenase activity: and
(iii) selecting the genetically modified lactic acid bacterium having a
reduction of at
least 25% of diacetyl reductase activity, acetoin reductase activity and
butanediol
dehydrogenase activity in the presence of NADH, NADPH, NAD+ or NADP+.

2. The bacterium according to claim 1 which is of Leuconostoc
psetrdomesenteroides.
3. The bacterium according to claim 2 which is selected from the group
consisting of
Leuconostoc pseudomesenteroides strain DSM 12099 and Leuconostoc
pseudomesenteroides
strain DSM 12465.

4. The bacterium according to claim 1 which is modified to have, in the
presence of NADH and
NADPH, a reduction of at least 25% of diacetyl reductase activity, acetoin
reductase activity and
butanediol dehydrogenase activity.

5. The bacterium according to claim 1 which is modified to have a reduction of
at least 90% of
diacetyl reductase activity, acetoin reductase activity and butanediol
dehydrogenase activity:

6. The bacterium according to claim 1 that, under cofactor conditions, where
the bacterium prior
to being modified has diacetyl reductase activity, acetoin reductase activity
and butanediol
dehydrogenase activity, is substantially incapable of said enzyme activities.


38
7. The bacterium according to claim 6 that, in the absence of NADPH,
substantially lacks
diacetyl reductase activity and acetoin reductase activity in a medium
containing NADH.

8. The bacterium according to claim 6 that, in the absence of NADH,
substantially lacks diacetyl
reductase activity and acetoin reductase activity in a medium containing
NADPH.

9. The bacterium according to claim 6 that substantially lacks diacetyl
reductase activity and
acetoin reductase activity in a medium containing both NADH and NADPH.

10. The bacterium according to claim 6 that, in the absence of NADP+,
substantially lacks
butanediol dehydrogenase activity in a medium containing NAD+.

11. The bacterium according to claim 6 that, in the absence of NAD+,
substantially lacks
butanediol dehydrogenase activity in a medium containing NADP+.

12. The bacterium according to claim 6 that substantially lacks butanediol
dehydrogenase
activity in a medium containing both NAD+ and NADP+.

13. The bacterium according to claim 1 that is obtained by the mutagenization
treatment of a
parent lactic acid bacterial strain which under appropriate cofactor
conditions has diacetyl
reductase activity, acetoin reductase activity and butanediol dehydrogenase
activity.

114. A starter culture composition comprising said genetically modified lactic
acid bacterium
according to claim 1 and a standard growth medium.

15. The composition according to claim 14 that is a frozen, dried or freeze-
dried composition.
16. The composition according to claim 15 containing a viable amount of said
genetically
modified lactic acid bacteria which is in the range of 10 4 to 10 12 cfu per
g.

17. The bacterium according to claim 13 that is obtained by contacting the
parent strain with a
chemical mutagen or UV light.

18. The bacterium according to claim 1 that is obtained by recombinant DNA
technology.


39
19. A method of preparing a fermented food product, the method comprising
adding the starter
culture composition according to any one of claims 14 to 16 or the genetically
modified lactic
acid bacterium according to any one of claims 1 to 13, 17 or 18 and at least
one acetaldehyde
producing strain of a lactic acid bacterial species to a food product starting
material wherein the
lactic acid bacterium or the composition has a reduction of at least 25% of
diacetyl reductase
activity, acetoin reductase activity and butanediol dehydrogenase activity and
keeping the
starting material under conditions where the starter culture composition is
capable of fermenting
said starting material to obtain the fermented food,

20. The method according to claim 19 wherein the resulting fermented food
product is a dairy
product selected from the group consisting of sour cream, cheese, fresh cheese
and buttermilk.
21. The method according to claim 19, wherein said product has an increased
content of
diacetyl of 10% (w/v), and at least 10% (w/v) of the diacetyl content is
retained after storage of
the product for 20 days or more at a temperature of about 4°C.

22. A food product obtained by the method according to any one of claims 19 to
21.

23. The food product according to claim 22 wherein the food product comprises
a bacterial
strain selected from the group consisting of DSM 12099 and DSM 12465.

24. The food product according to claim 23 which is selected from the group
consisting of sour
cream, cheese, fresh cheese and buttermilk.

Note: Descriptions are shown in the official language in which they were submitted.


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NOVEL GENETICALLY MODIFIED LACTIC ACID BACTERIA HAVING MODIFIED
DIACETYL REDUCTASE ACTIVITIES

FIELD OF THE INVENTION

The present invention relates to the field of manufacturing food products by
means of
lactic acid bacterial cultures. Specifically the invention provides novel
genetically mo-
dified strains of lactic acid bacteria that are modified to have enhanced or
reduced
diacetyl reductase activity, acetoin reductase activity and/or butanediol
dehydrogenase
activity. Such modified bacteria are particularly useful in the manufacturing
of food
products having either a reduced or an increased content of the flavour
compound
diacetyl.

TECHNICAL BACKGROUND AND PRIOR ART

Lactic acid bacteria are used extensively as starter cultures in the food
industry in the
manufacturing of fermented products including milk products such as e.g.
yoghurt and
cheese, meat products, bakery products, wine and vegetable products.
Lactococcus
species including Lactococcus lactis are among the most commonly used lactic
acid
bacteria in dairy starter cultures. Several other lactic acid bacteria such as
Leuconostoc species, Pediococcus species, Lactobacillus species, Oenococcus
species
and Streptococcus species are also commonly used in food starter cultures.
When a lactic acid bacterial starter culture is added to milk or any other
food product
starting material under appropriate conditions, the bacteria grow rapidly with
concomitant conversion of citrate, lactose or other sugar compounds into
lactic
acid/lactate and possibly other acids including acetate, resulting in a pH
decrease. In
addition, several other metabolites are produced during the growth of lactic
acid
bacteria. These metabolites include ethanol, formate, acetaldehyde, a-
acetolactate,
acetoin, diacetyl, carbon dioxide and 2,3 butylene glycol (butanediol).


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2

Among these metabolites, diacetyl (2,3-butanedione) is an essential flavour
compound
in dairy products such as butter, yoghurt, starter distillate, margarine,
buttermilk and
cheese. However, its presence in other products, such as fruit juices, beers
and
wines, is undesirable, as it imparts a buttery or toffee taste and is the
agent
responsible for the so-called sarcina sickness of beer. The compound is formed
during
fermentation of lactic acid bacterial species of e.g. Lactococcus, Leuconostoc
and
Lactobacillus by an oxidative decarboxylation of a-acetolactate which is
formed from
two molecules of pyruvate by the action of a-acetolactate synthase (ALS).

Diacetyl reducing enzymes, commonly termed diacetyl reductases (DR)
(acetoin:NAD
oxidoreductases E.C. 1.1.1.5), have been observed from many different sources,
notably animal tissues (Provecho et at., 1984), bacteria including Lactococcus
(formerly Streptococcus) lactis (Crow, 1990; Arora et al., 1978), Bacillus
species and
Enterobacter species (Giovannini et al. 1996), and yeast (Gibson et al.,
1991).
Boumerdassi et al. 1997 disclosed a mutated Lactococcus lactis strain having
DR
activity that was increased by three times relative to the activity of the
parent strain.
In Arora et al. 1978 and Kulia & Ranganathan 1978 are disclosed mutants of
Lactococcus lactis having a reduced diacetyl activity when grown in non fat
dry milk
and citrate medium, respectively.
Generally, the term "diacetyl reductase" ("DR") encompasses several enzymatic
activities such as diacetyl reductase activity, acetoin reductase activity
and/or
butanediol dehydrogenase activity which carry out the following enzymatic
reactions;
diacetyl + NAD(P)H ---> acetoin + NAD(P)+, acetoin + NAD(P)H <---> butanediol
+ NAD(P)+, respectively. Thus, L. lactis has been reported to possess two
diacetyl
reductases with activity for both diacetyl and acetoin. Both of these enzymes
use
NADH as cofactor (Crow, 1990).

Leuconostoc species including Leu. pseudomesenteroides are typically used in
mixed
starter cultures together with Lactococcus lactis subsp. lactis and
Lactococcus lactis
subsp. lactis biovar. diacetylactis in the production of dairy products. A
significant role
of Leuconostoc species in such mixed cultures is to remove the acetaldehyde
produced by the accompanying strains e.g. in the production of buttermilk and
fresh
cheeses. However, Leuconostoc strains will also remove diacetyl by reducing it
into


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3

acetoin and/or butanediol, a characteristic that is generally undesirable in
the produc-
tion of dairy products. The enzyme responsible for the reduction of diacetyl,
diacetyl
reductase, is highly expressed in Leuconostoc species such as Leu.
pseudomesente-
roides which species is known to have about 100 times higher diacetyl
reductase
activity than L. lactis.

Thus, one primary objective of the present invention is to provide lactic acid
bacterial
cultures of species, including Leuconostoc species, that inherently have one
or more
DR activities which, relative to the naturally occurring strains, has reduced
or
substantially eliminated DR activities under specific cofactor conditions. By
providing
such strains to the industry, it has become possible to produce lactic acid
bacterial
fermented food products having a desirably high content of diacetyl.

Another objective of the invention is to provide lactic acid bacterial strain
that, relative
to the presently available strains, has a strongly enhanced DR activities.
Using such
strains which utilise diacetyl as a substrate it is possible to reduce or
remove diacetyl
in food products where the presence of this flavour compound is undesirable.

SUMMARY OF THE INVENTION

Accordingly, the invention provides in a first aspect a genetically modified
lactic acid
bacterium, including the Leuconostoc pseudomesenteroides strains DSM 12099 and
DSM 12465 and lactic acid bacteria essentially having the diacetyl reductase
characteristics of these strains, that, relative to the lactic acid bacterium
from which it
is derived, is modified to have a reduction of at least one of diacetyl
reductase
activity, acetoin reductase activity and butanediol dehydrogenase activity,
said
modified bacterium,

(i) is substantially incapable of at least one of diacetyl reductase activity
and acetoin
reductase activity in a medium containing NADH and not containing NADPH, or

(ii) is substantially incapable of at least one of diacetyl reductase activity
and acetoin
reductase activity in a medium containing NADPH and not containing NADH, or


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4

(iii) is substantially incapable of at least one of diacetyl reductase
activity and acetoin
reductase activity in a medium containing both NADH and NADPH, or

(iv) is substantially incapable of butanediol dehydrogenase activity in a
medium
containing NAD+ and not containing NADP+, or

(v) is substantially incapable of butanediol dehydrogenase activity in a
medium
containing NADP+ and not containing NAD+, or
NO is substantially incapable of butanediol dehydrogenase activity in a medium
containing both NAD+ and NADP+,

where the bacterium prior to being modified is capable of having at least one
of said
enzymatic activities under said cofactor conditions.

In a further aspect, the invention relates to a genetically modified lactic
acid bacterium
that, relative to the lactic acid bacterium from which it is derived, is
modified to have
a reduction of at least one of diacetyl reductase activity, acetoin reductase
activity
and butanediol dehydrogenase activity, including the Leuconostoc
pseudomesentero-
ides strains DSM 12099 and DSM 12465 and lactic acid bacteria essentially
having
the diacetyl reductase characteristics of these strains, subject to the
limitation, that
the lactic acid bacterium is not Lactococcus lactis.

In a still further aspect, the invention relates to a genetically modified
lactic acid
bacterium that has no detectable diacetyl reductase activity, acetoin
reductase activity
and/or butanediol dehydrogenase activity, subject to the limitation, that the
lactic acid
bacterium is not Lactococcus lactis.

In other further aspects, the invention relates to a genetically modified
lactic acid
bacterium that, relative to the lactic acid bacterium from which it is
derived, is
modified to have an enhancement of at least one of diacetyl reductase
activity,
acetoin reductase activity and butanediol dehydrogenase activity which is at
least 10


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times, including the Lactococcus lactis subsp. lactis strain DSM 12096 and
lactic acid
bacteria essentially having the diacetyl reductase characteristics of that
strain.

In a still further aspect, the invention pertains to a starter culture
composition
5 comprising such a genetically modified bacterium.

There is also provided a method of preparing a fermented food product,
comprising
adding an effective amount of a bacterium that, relative to the lactic acid
bacterium
from which it is derived, is modified to have a reduction of at least one of
diacetyl
reductase activity, acetoin reductase activity and butanediol dehydrogenase
activity,
or a composition comprising such a bacterium to a food product starting
material
wherein the bacterium or the composition is incapable of having at least one
enzymatic activity selected from the group consisting of diacetyl reductase
activity,
acetoin reductase activity and butanediol dehydrogenase activity and keeping
the
starting material under conditions where the bacterium or the starter culture
composition is capable of fermenting said starting material to obtain the
fermented
food, and a fermented food product obtainable by such a method which product
has a
content of diacetyl which is at least 10% higher than that of a product
fermented
under identical conditions with a parent strain for the genetically modified
bacterium.
In yet another aspect, the invention relates to a method of producing a food
product,
comprising adding an effective amount of a bacterium that, relative to the
lactic acid
bacterium from which it is derived, is modified to have an enhancement of at
least one
of diacetyl reductase activity, acetoin reductase activity and butanediol
dehydrogenase
activity, or a composition comprising such a bacterium to a food product
starting
material that contains at least one of diacetyl, acetoin and butanediol, and
keeping the
starting material under conditions where the genetically modified lactic acid
bacterium
has at least one enzymatic activity selected from the'group consisting of
diacetyl
reductase activity, acetoin reductase activity and butanediol dehydrogenase
activity to
obtain a product having a reduced content of diacetyl.


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6

DETAILED DISCLOSURE OF THE INVENTION

It is, as it is mentioned above, an important objective of the present
invention to
provide lactic acid bacteria that has a reduced capability to convert diacetyl
in a
fermented food product to acetoin and/or butanediol. Accordingly, in one
aspect the
genetically modified lactic acid bacterium is a bacterium that, relative to
the lactic acid
bacterium from which it is derived, is modified so as to have a reduction of
at least
one of diacetyl reductase activity, acetoin reductase activity and butanediol
dehydrogenase activity, said bacterium, when grown under at least one of the
above
cofactor conditions, where the bacterium prior to being mutated is capable of
having
at least one of said enzymatic activities, is substantially incapable of at
least one of
said activities. As used herein, the term "substantially incapable" indicates
that the
respective enzymatic activities can not be detected by the assay procedures
described
herein.
As used herein, the expression "lactic acid bacterium" refers to a group of
gram-
positive, microaerophilic or anaerobic bacteria having in common the ability
to ferment
sugars and citrate with the production of acids including lactic acid as the
predominantly produced acid, acetic acid, formic acid and propionic acid. The
industrially most useful lactic acid bacteria are found among Lactococcus
species,
Streptococcus species, Lactobacillus species, Leuconostoc species, Oenococcus
species and Pediococcus species. In the dairy industry, the strict anaerobes
belonging
to the genus Bifidobacterium is generally included in the group of lactic acid
bacteria
as these organisms also produce lactic acid and are used as starter cultures
in the
production of dairy products.

It will be appreciated that the term "genetically modified" as used herein
indicates any
modification of DNA sequences coding for genes involved in the expression of
DR
activities including modifications of sequences that regulate the expression
of genes
coding for such enzymatic activities. Accordingly, genetic modification can be
based
on construction or selection of mutants of lactic acid bacteria or it can be
based on
recombinant DNA-technology. When the term "diacetyl reductase" or "DR" is used
herein it refers to any of the three mentioned specific activities, i.e.
diacetyl reductase
activity, acetoin reductase activity and butanediol dehydrogenase activity.


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7

As used herein the term "mutant" is used in the conventional meaning of that
term i.e.
it refers to strains obtained by subjecting a lactic acid bacterial strain to
any conven-
tionally used mutagenization treatment including treatment with a chemical
mutagen
such as ethanemethane suiphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine
(NTG),
UV light or to spontaneously occurring mutants which are selected on the basis
of a
modified DR activity. Although it is presently preferred to provide the
genetically
modified bacteria according to the invention by random mutagenesis or by
selection of
spontaneously occurring mutants, i.e. without the use of recombinant DNA-
technology, it is envisaged that mutants of lactic acid bacteria can be
provided by
such technology including site-directed mutagenesis and PCR techniques and
other in
vitro or in vivo modifications of DNA sequences coding for DR activities or
sequences
regulating the expression of genes coding for the DR activities, once such
sequences
have been identified and isolated.
It is also possible to construct genetically modified bacteria according to
the invention
by conventional recombinant DNA-technology including insertion of sequences
coding
for DR activities, e.g. by replacing a native promoter for such coding
sequences by a
foreign promoter which either enhances or reduces the expression of the coding
sequences. It is also possible to derive lactic acid bacterial strains
according to the
invention from species that do not have an inherent capability to produce DR
activities
by inserting genes coding for such activities isolated from a different
organism
comprising such genes. The source of such genes may be bacterial species,
yeast
species or mammal species. Additionally, it is envisaged that genetically
modified
bacteria according to the invention can be constructed by modifying metabolic
pathways in a lactic acid bacterium that are not directly involved in DR
pathways.
It will be appreciated that the expression "under cofactor conditions" as used
herein
indicates the absence/presence in an appropriate medium of any non-protein
substance required for biological activity of any of the enzyme activities
according to
the invention, such as NAD+, NADH, NADP+ and NADPH.

A genetically modified bacterium having a reduced diacetyl activity can be
selected
from any kind of lactic acid bacterial species which has an inherent DR
activity,
including Lactococcus spp., Streptococcus spp., Lactobacillus spp.,
Leuconostoc spp


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8

such as Leuconostoc pseudomesenteroides, Pediococcus spp., Oenococcus spp. and
Bifidobacterium spp.

As mentioned above, the invention relates in another aspect to a genetically
modified
lactic acid bacterium that, relative to the lactic acid bacterium from which
it is derived,
is modified to have a reduction of at least one of diacetyl reductase
activity, acetoin
reductase activity and butanediol dehydrogenase activity, including the
Leuconostoc
pseudomesenteroides strains DSM 12099 and DSM 12465 and lactic acid bacteria
essentially having the diacetyl reductase characteristics of these strains,
subject to the
limitation that the modified bacterium is not Lactococcus lactis.

However, in particularly useful embodiments, the above genetically modified
bacterium
is one that under cofactor conditions, where the bacterium prior to being
genetically
modified is capable of having at least one of diacetyl reductase activity,
acetoin
reductase activity and butanediol dehydrogenase activity, is substantially
incapable of
at least one of said enzymatic activities.

Such a bacterium includes a bacterium that is substantially incapable of at
least one of
diacetyl reductase activity and acetoin reductase activity in a medium
containing
NADH and not containing NADPH, a bacterium that is substantially incapable of
at
least one of diacetyl reductase activity and acetoin reductase activity in a
medium
containing NADPH and not containing NADH, a bacterium that is substantially
incapable of at least one of diacetyl reductase activity and acetoin reductase
activity
in a medium containing both NADH and NADPH, a bacterium that is substantially
incapable of butanediol dehydrogenase activity in a medium containing NAD+ and
not
containing NADP, a bacterium that is substantially incapable of butanediol
dehydrogenase activity in a medium containing NADP+ and not containing NAD+
and
a bacterium that is substantially incapable of butanediol dehydrogenase
activity in a
medium containing both NAD+ and NADP+.

In a further aspect, the invention provides a genetically modified lactic acid
bacterium
that has no detectable diacetyl reductase activity, acetoin reductase activity
and/or
butanediol dehydrogenase activity. Such a bacterium is selected from any of
the


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9

above-mentioned lactic acid bacterial species, subject to the limitation, that
the
bacterium is not Lactococcus lactis.

A genetically modified bacterium having reduced or no detectable DR activities
can be
derived from any lactic acid bacterial species which has an inherent DR
activity,
including Lactococcus spp. such as Lactococcus lactis subsp. lactis biovar.
diace-
tylactis and Lactococcus lactis subsp. lactis, Streptococcus spp. including
Streptococcus thermophilus, Lactobaci//us spp., Leuconostoc spp. including
Leuconostoc pseudomesenteroides., Pediococcus spp.. Oenococcus spp. and Bifi-
dobacterium spp.

Although it may be preferred that the modified bacterium has substantially no
detectable DR activities, a bacterium that is modified to have a reduction of
one or
more of the above activities is also encompassed by the invention. Thus, a
useful
bacterium according to the invention is one that has a reduction in any of the
DR
activities which, relative to the bacterium from which it is derived, is at
least 25%
such as at least 50% including at least 75% e.g. at least 90%. Thus, the
genetically
modified bacterium according to the invention preferably has a DR activity
reduction
which is reduced by at least 25 times for anyone of the enzymatic activities
such as
at least 50 times, including at least 100 times or even at least 500 times,
relative to
the strain from which it is derived.

When a modified lactic acid bacterial strain according to the invention is
added to a
food product starting material, such as e.g. milk, wherein the bacterium is
incapable of
having at least one of the above DR enzymatic activities and the starting
material is
kept under conditions where the strain is capable of fermenting said starting
material
to obtain a fermented food product, the resulting food product preferably has
an
increased content of diacetyl which is at least 1.1 times higher, such as at
least 2
times higher, including at least 5 times higher or even at least 10 times
higher, relative
to a similar food product which is fermented using the strain from which the
modified
strain is derived.

Thus in one embodiment, the modified bacterium according to the invention is
derived
by subjecting a parent lactic acid bacterial strain that under appropriate
cofactor


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conditions is capable of having diacetyl reductase activity, acetoin reductase
activity
and/or butanediol dehydrogenase activity to a mutagenization treatment and
selecting
a strain that is substantially incapable of at least one of said enzymatic
activities under
identical cofactor conditions.
5
The present invention relates in a further aspect to a genetically modified
lactic acid
bacterium that, relative to the lactic acid bacterium from which it is
derived, is
modified to have an enhancement of at least one of diacetyl reductase
activity,
acetoin reductase activity and butanediol dehydrogenase activity which is at
least 10
10 times, including the Lactococcus lactis subsp. lactis strain DSM 12096 and
lactic acid
bacteria essentially having the diacetyl reductase characteristics of that
strain.

It was found that it is possible to provide genetically modified lactic acid
bacteria that
have a significant enhancement of the specific DR activities. Thus, by
fermenting a
material or a medium having a content of diacetyl with such a genetically
modified
bacterium it is possible to obtain a final product wherein essentially all of
the diacetyl
has been converted to butanediol which is without the buttery flavour of
diacetyl.
Thus, the genetically modified bacterium according to the invention preferably
has an
activity enhancement which is at least 10 times for anyone of the enzymatic
activities
such as at least 50 times or even at least 100 times, relative to the strain
from which
it is derived.

A genetically modified bacterium having enhanced DR activities can be derived
from
any lactic acid bacterial species which has an inherent DR activity, including
Lactococcus spp. such as Lactococcus lactis subsp. lactis biovar.
diacetylactis and
Lactococcus lactis subsp. lactis, Streptococcus spp. including Streptococcus
thermophi/us, Lactobacillus spp., Leuconostoc spp. including Leuconostoc
pseudomesenteroides., Pediococcus spp. and Bifidobacterium spp.

It will be appreciated that such modified bacteria can be a spontaneous mutant
or be
provided by subjecting a lactic acid bacterium that has inherent DR activities
to a
mutagenization treatment as described above or by inactivating or deleting one
or
more genes involved in the expression of the DR activities using conventional
recombinant DNA-technology.


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The genetically modified bacteria according to the invention are useful as
starter
cultures in the production of food products. Accordingly, in a further
important aspect,
the invention relates to a starter culture composition comprising a bacterium
according
to the invention either having enhanced or a reduced or eliminated DR
activities.
Typically, such a composition comprises the bacteria in a concentrated form
including
frozen, dried or freeze-dried concentrates typically having a concentration of
viable
cells which is in the range of 104 to 1012 cfu per g including at least 104
cfu per gram
of the composition, such as at least 105 cfu/g, e.g. at least 106 cfu/g, such
as at least
10' cfu/g, e.g. at least 106 cfu/g, such as at least 109 cfu/g, e.g. at least
1010 cfu/g,
such as at least 1011 cfu/g of the composition. The composition may as further
components contain cryoprotectants and/or conventional additives including
nutrients
such as yeast extract, sugars and vitamins.
As it is normal in the production of lactic acid bacterial fermentation
processes to
apply mixed cultures lactic acid bacteria, the composition will in certain
embodiments
comprise a multiplicity of strains either belonging to the same species or
belonging to
different species. A typical example of such a useful combination of lactic
acid
bacteria in a starter culture composition is a mixture of a Leuconostoc spp.
and one or
more Lactococcus spp. such as Lactococcus lactis subsp. lactis or Lactococcus
lactis
subsp. lactis biovar. diacetylactis. Such a mixed culture can be used in the
manu-
facturing of fermented milk products such as buttermilk and cheese. It will be
understood that in such a mixed culture of lactic acid bacteria, one or more
of the
strain components may be a modified bacterium according to the invention.

It is also an objective of the invention to provide a method of preparing a
fermented
food product based on the use of the genetically modified bacteria of the
invention
which have reduced or lacking DR activities. In its broadest aspect, such a
method
comprises that an effective amount of such bacteria or a composition
comprising the
bacteria are added to a food product starting material wherein the bacterium
or the
composition is incapable of having at least one of the above DR enzymatic
activities
and keeping the starting material under conditions where the bacterium or the
starter


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culture composition is capable of fermenting said starting material to obtain
a
fermented food product.

Useful food product starting materials include any material which is
conventionally
subjected to a lactic acid bacterial fermentation step such as milk, vegetable
materials,
meat products, fruit juices, must, doughs and batters. The fermented products
which
are obtained by the method include as typical examples dairy products such as
cheese
including fresh cheese products, and buttermilk.

As it is mentioned above, the use in food starter cultures of bacteria
according to the
invention that have a reduced or lacking DR activity will result in final
products having
a content of the desired flavour compound diacetyl which is higher than would
otherwise be obtained if a non-modified lactic acid bacterium was used.
Accordingly,
it is an important aspect of the invention to provide a fermented food product
obtainable by the above method which product has a content of diacetyl which
is at
least 10% higher such as at least 20% higher or at least 30% higher than that
of a
product fermented under identical conditions with a parent strain for the
genetically
modified bacterium. Examples of such food products include milk-based products
such
as cheese and buttermilk, vegetable products, meat products, fruit juices,
wines and
bakery products.

As shown in the below Examples, when the DR mutant MM084 is used as a
component of a mixed flavour-forming starter culture for the fermentation of
one of
the above starting materials, the mutant has a significant effect on the
diacetyl
stability during storage of the resulting fermented product.

Thus, an advantageous feature of the fermented food product according to the
invention is that the food product can be stored for several weeks with less
reduction
in the diacetyl content than is the case with a food product fermented under
identical
conditions with the parent strain of the genetically modified bacterium. Thus,
in one
particularly useful embodiment, the fermented product is one which at least
10% of
its initial diacetyl content is retained after storage for 20 days or more at
about 4 C.
when stored under appropriate storage conditions, such as at least about 20%
of its
initial diacetyl content e.g. at least about 30% and preferably at least about
40% e.g.


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at least about 50% of its initial diacetyl content is retained after storage
for 20 days
or more at about 4 C. This improvement implies that a fermented food product
manufactured by use of the above mixed starter culture can be stored for an
extended
period of time without loosing its desired flavour.
Whereas in many lactic acid bacterial fermented food products it is desirable
to have a
high content of diacetyl, this may be undesirable in other products. This is
in particular
the case in beverages such as fruit juices, beers and other yeast fermented
beverages
including certain wines, where diacetyl imparts to the products a buttery or
toffee
taste. In beers a diacetyl content above the threshold level gives rise to the
so-called
sarcina sickness. It is therefore an interesting aspect of the invention to
provide a
method of producing a food product having a reduced content of diacetyl.

This method comprises adding an effective amount of a lactic acid bacterium
that has
been modified to have at least one increased DR activity or a composition
containing
such a bacterium to a food product starting material that contains at least
one of
diacetyl, acetoin and butanediol, and keeping the starting material under
conditions
where the genetically modified lactic acid bacterium has at least one
enzymatic
activity selected from the group consisting of diacetyl reductase activity,
acetoin
reductase activity and butanediol dehydrogenase activity to obtain a product
having a
reduced content of diacetyl. In useful embodiments the products resulting from
such a
method have no detectable content of diacetyl.

The invention will now be described in further details in the following non-
limiting
examples and the drawings wherein

Fig. 1. shows native-PAGE gels containing cell free extracts of wild-type
strain of Leu.
pseudomesenteroides DB1 334. The gels were incubated with diacetyl + NADH (A);
butanediol + NAD+ (B); and acetoin + NADH (C), and stained with Meldola's blue
and MTT. 2, 4, and 8 gg of protein, respectively were loaded onto each gel;

Fig. 2 shows native-PAGE gels containing cell free extracts of wild-type
strain of Leu.
pseudomesenteroides DB1 334 and mutant strain MM084 stained with the zymogram
technique. The gels were incubated with diacetyl + NADPH (A) or butanediol +


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NADP+ (B). MM084 was loaded in lanes 1-4 and DB1334 in lanes 5, 7 and 9.
Approximately 6 lag of protein was loaded in each lane;

Fig. 3 illustrates the diacetyl content in reconstituted skimmed milk
fermented by the
mixed cultures A and B during fermentation and storage. The reconstituted
skimmed
milk was fermented at 22 C in non-shaken bottles and subsequently stored at 4
C,
and

Fig. 4 illustrates the diacetyl content in sour cream fermented with the mixed
cultures
C and D during fermentation and storage. The cream was fermented at 22 C and
subsequently stored at 4 C.

EXAMPLE 1
Construction of a NADH-dependent diacetyl reductase mutant of Leuconostoc
pseudomesenteroides

1.1. Summary of experiments
Partially purified NADH-dependent diacetyl reductase from Leuconostoc
pseudomesenteroides showed that the enzyme is responsible for at least three
enzymatic reactions: (i) diacetyl + NADH ---> acetoin + NAD+; (ii) acetoin +
NADH
---> butanediol + NAD+; and (iii) butanediol + NAD+ ---> acetoin + NADH. The
enzymatic properties of diacetyl reductase were demonstrated by staining
native
PAGE gels using the zymogram technique as described in the following. Using
this
technique, the immobilised enzyme is allowed to react with a substrate and
cofactor
with a subsequent dye staining. The same technique was also used to screen an
ethanemethane sulphonate (EMS) mutagenized Leu. pseudomesenteroides population
for the absence of diacetyl reductase (butanediol dehydrogenase activity)
activity by
direct staining of colonies immobilised onto nitrocellulose membranes. Enzyme
activity
measurements from cell extracts showed that the mutant possessed only minute
diacetyl reductase activity, no acetoin reductase or butanediol dehydrogenase
activity
when reacting with NADH or NAD+. However, activities comparable to the wild-
type


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strain were obtained when using NADPH or NADP+ as cofactors, indicating the
presence of two distinct diacetyl reductases in Leu. pseudomesenteroides.

In this example, the isolation and characterisation of a mutant strain of Leu.
5 pseudomesenteroides that is defective with respect to an NADH-dependent
diacetyl
reductase is described.

1.2. Materials and Methods
10 (i) Bacterial strain

A wild-type strain of Leu. pseudomesenteroides DB1334 (Chr. Hansen Culture
Collection (CHCC) 21 14) was used in the experiment.

15 (ii) Cultivation conditions

Leu. pseudomesenteroides was cultivated in M 17 medium (Terzaghi & Sandine,
1975)
supplemented with 0.5% glucose at 25 C under anaerobic conditions.

(iii) Mutagenesis

Leu. pseudomesenteroides was cultivated in 10 ml M17 (0.5% glucose) for 3 days
followed by cultivation for 120 minutes in the presence of 150 lal of EMS.
After EMS
treatment, 0.2 ml of the culture was inoculated into ten tubes each containing
10 ml
of M 17 and incubated for 3 days for phenotypic expression. The mutation
frequency
was monitored by plating 0.1 ml from each tube onto M17 plates containing 500
4g/ml of streptomycin.

(iv) Colony screening for mutants

Cells having been subjected to mutagenization were plated on M17 (0.5%
glucose)
and incubated anaerobically for 2 days at 25 C and subsequently streaked onto
duplicate M17 plates. After another 2 days of incubation one of the duplicate
plates
was used for screening. The colonies were transferred onto a nitrocellulose
membrane


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and soaked for 1.5 minutes in chloroform for cell lysis. After cell lysis, the
membrane
was washed with distilled water and dried for 20 minutes. The membrane was
next
incubated for 30 minutes in a solution containing 0.5 M Na-phosphate buffer
(pH 6.1),
72 mM butanediol, 1 mM NAD+, 0.02 mM Meldola's blue (8-Dimethylamino-2,3-
benzophenoxazine) 0.8 mM MTT (3-[4,5-Dimethylthiazol-2-yl] 2,5-
diphenyltetrazolium
bromide; Thiazolyl blue).

(v) Protein electrophoresis

Using 4-20% Tris-HCI gradient gels with Tris-Glycine (pH 8.3) as running
buffer,
native-PAGE was run at 150 V for 2.5 hours. Staining of native gels was
performed
both with 0.25% Coomassie brilliant blue in 10% acetic acid and 40% methanol
and
with the zymogram technique (see below). SDS-PAGE was run using a 12%
separation gel, 4% stacking gel and with Tris-Glycine (pH 8.3) as running
buffer at
200 V for 45 minutes.

(vi) Zymogram staining of gels

Zymogram staining of native-PAGE gels for identification of diacetyl reductase
activity
and butanediol dehydrogenase activity was performed as follows: for diacetyl
reductase activity the gel was incubated for 15 minutes with 12 mM diacetyl,
1.5 mM
NADH, 0.5 M Na-phosphate buffer (pH 6.1) and for butanediol dehydrogenase
activity
the gel was incubated for 15 minutes with 72 mM butanediol, 1 mM NAD+, 0.5 M
Na-phosphate buffer. The gel was subsequently incubated for 30 minutes under
dry
conditions before the addition of a solution consisting of 0.02 mM Meldola's
blue (8-
Dimethylamino-2, 3-benzophenoxazine) 0.8 mM MTT (3-[4,5-Dimethylthiazol-2-yl]
2,5-
diphenyltetrazolium bromide; Thiazolyl blue) in 100 mM phosphate buffer (pH
8.2)
(Provecho et al, 1984; Gibson et al, 1991). Visible bands appeared within 20
minutes.
(vii) Preparation of cell free extracts

Leu. pseudomesenteroides was cultivated in M 17 (0.5 % glucose). The cells
were
harvested in the exponential growth phase by centrifugation at 6000 rpm for 15
minutes and washed in cold 50 mM Na-phosphate buffer (pH 6). The pellet was


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resuspended in cold 50 mM Na-phosphate buffer (pH 6) and sonicated for 3 x 2
minutes. The sonicated cell mixture was centrifuged at 6000 rpm for 15 min,
and the
supernatant was stored at -20 C until analysed for protein content and enzymic
activities.
(viii) Enzyme activity measurements

Diacetyl reductase activity was measured spectrophotometrically by monitoring
the
oxidation of NADH at 340 nm in a reaction mixture with the following
composition:
50 mM Na-phosphate buffer (pH 6.1), 36 mM diacetyl and 0.5 mM NADH. Butanediol
dehydrogenase activity was measured by monitoring the reduction of NAD+ at 340
nm in a reaction mixture with the following composition: 50 mM phosphate
buffer (pH
6.1), 72 mM butanediol and 1 mM NAD+. Acetoin reductase activity was measured
by monitoring the oxidation of NADH at 340 nm in a reaction mixture with the
following composition: 50 mM Na-phosphate buffer (pH 6.1), 36 mM acetoin and
0.5
mM NADH.

Lactate dehydrogenase was measured by monitoring the oxidation of NADH at 340
nm in a reaction mixture with the following composition: 50 mM Tris-acetate
buffer
(pH 6), 0.5 mM fructose-l,6-diphosphate, 25 mM pyruvate and 0.5 mM NADH.

The specific enzymatic activities were expressed as micromoles of converted
substrate per milligram of protein per minute (equivalent to units per
milligram protein).
(ix) Protein determination

Protein content was measured by using the BCA Protein Assay Reagent (Pierce)
with
bovine serum albumin as the standard.

(x) Milk fermentation

Boiled (UB) 9.5% reconstituted skim-milk (RSM) supplemented with 100 ppm of
acetaldehyde and 100 ppm of diacetyl was used as fermentation substrate. The
milk


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was inoculated with DB1 334 and MW008 and samples were analysed by headspace
GC every hour for twenty hours (Richelieu et al., 1997).

1.3. Results
(i) Native PAGE staining

Partially purified diacetyl reductase from Leu. pseudomesenteroides was
separated on
native gradient PAGE gels and stained with both Coomassie brilliant blue and
with the
zymogram technique. With the zymogram technique, the gels were incubated with
diacetyl + NADH, acetoin + NADH or with butanediol + NAD+. The gels were
subsequently stained with Meldola's blue and MTT. With the three different
incubation
mixtures a protein band with the same molecular weight was visualised. In the
presence of diacetyl + NADH, or with acetoin + NADH, the gel becomes saturated
with NADH. However, at the position where diacetyl reductase is immobilised,
the
enzyme converts diacetyl + NADH into acetoin + NAD+ or acetoin + NADH into
butanediol +NAD+. In the following incubation with Meldola's blue and MTT,
these
reagents react with the reduced cofactor (NADH) and the gel becomes purple
except
where diacetyl reductase is located. The band corresponding to diacetyl
reductase
becomes colourless. Incubation with butanediol and NAD+ results in the reverse
result.
In this case, butanediol + NAD+ is converted to acetoin + NADH resulting in a
purple
band with a colourless background. No reaction was observed with acetoin +
NAD+.
Fig. 1 shows a native-PAGE gel incubated with different substrates and
cofactors
followed by staining with MTT and Meldola's blue.
(ii) Screening of EMS mutagenized DB1334

Based on the result from the zymogram staining, an EMS mutagenized DB1 334
population was screened by incubating the cells in a reaction mixture of
butanediol +
NAD+. Lysed cells with an intact diacetyl reductase (butanediol dehydrogenase
activity) were stained purple whereas a diacetyl reductase (DR) mutant should
become
colourless. Approximately 1700 clones were screened and 1 clone appeared
colourless. This putative DR mutant was restreaked three times and repeatedly
stained


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with Meldola's blue and MTT before regarded as true mutant. The selected
mutant
was designated MW008.

A sample of the mutant Leu. pseudomesenteroides MW008 strain was deposited in
accordance with the Budapest Treaty with the Deutsche Sammlung vor
Mikroorganismen and Zellkulturen (DSMZ), Marschenroder Weg, 1 b, D-381 24
Braunschweig on 7 April 1998 under the Accession No. DSM 12099.

(iii) Enzyme activity measurements
Cell-free extracts of the DR mutant (MW008) and DB1 334 were used for
measuring
the diacetyl reductase, butanediol dehydrogenase, acetoin reductase and
lactate
dehydrogenase activities. Lactate dehydrogenase activity measurements were
used as
a positive control for enzymatic activity of the strains. The results of the
enzyme
activities are summarised in Table 1.1.

Table 1.1. Diacetyl reductase (DR), acetoin reductase (AR), butanediol
dehydrogenase
(BUTDH) and lactate dehydrogenase (LDH) activities from cell free extracts of
DB1 334
and MW008.

strain specific activity in U/mg of protein
DR' AR' BUTDH2 LDH'
DB1334 2.81 0.81 0.34 17.70
MW008 0.017 n.d n.d 22.10
' with NADH, 2 with NAD+
n.d = not detectable, activities below the detection limit <0.005 U/mg.
1.4. Conclusions

Based on the zymogram staining of native PAGE gels containing partially
purified
diacetyl reductase derived from Leu. pseudomesenteroides it was concluded that
this
enzyme has activity for diacetyl + NADH, acetoin + NADH, and butanediol +
NAD+.
No activity was observed with acetoin and NAD+. Screening of a mutagenized
population of Leu. pseudomesenteroides was performed by incubating the cells
with


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butanediol and NAD+. After staining, cells with an intact DR were stained
purple,
whereas a mutant became colourless. Cell-free extract from the DR mutant
confirmed
the result from the screening procedure, since essentially no activity was
found with
diacetyl + NADH, acetoin + NADH or butanediol + NAD+.
5
After isolation of the NADH-dependent DR mutant, this strain was used for
fermentation of milk supplemented with diacetyl and acetaldehyde.
Surprisingly, the
mutant strain was able to reduce diacetyl despite the absence of NADH-
dependent
diacetyl reductase activity. Measurements of cell free extract of the mutant
with
10 diacetyl + NADPH, acetoin + NADPH and butanediol + NADP+ showed similar
acti-
vities as the wild type. Therefore, it was most likely that DB1 334 has two
diacetyl
reductases responsible for diacetyl degradation. In order to prevent diacetyl
reduction
during milk fermentation, also the NADPH-dependent diacetyl reductase of DB1
334
must be mutated.

EXAMPLE 2

Demonstration of both NADH- and NADPH-dependent diacetyl reductase activities
in
Leuconostoc pseudomesenteroides and construction of mutant strains totally
blocked
in diacetyl reductase activities

2.1. Summary of experiments

In Example 1 the construction of a diacetyl reductase mutant with no
essentially
activity for diacetyl + NADH is described. However, the mutant possessed
diacetyl
reductase activities as the wild-type strain when using NADPH as cofactor.
This strain
was able to degrade diacetyl at the same rate as the wild-type strain. The
NADH-
dependent diacetyl reductase mutant was subjected to further mutagenization
and
screened for mutants incapable of reducing diacetyl both in the presence of
NADH and
NADPH as cofactors.


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2.2. Materials and Methods
(i) Bacterial strains

Leuconostoc pseudomesenteroides D131 334 (CHCC2114), MW008 (NADH-dependent
diacetyl reductase mutant, see Example 1) and MM084 (NADH-, NADPH-dependent
diacetyl reductase mutant, this Example).

(ii) Cultivation conditions
D131 334, MW008 and MM084 were cultivated on M17 (0.5% glucose) plates, or in
liquid medium, at 25 C under anaerobic conditions.

(iii) Mutagenesis
MW008 was cultivated in 10 ml M17 (0.5% glucose) for three days followed by
cultivation for 120 minutes in the presence of 150 I of EMS. After EMS
treatment,
0.2 ml of the culture was inoculated into ten tubes each containing 10 ml of M
17 and
incubated for 3 days for phenotypic expression. The mutation frequency was
monitored by plating 0.1 ml from each tube on M 17 plates containing 500 g/ml
of
streptomycin.

(iv) Colony screening

Mutated cells were plated on M17 (0.5% glucose) and incubated for 2 days at 25
C
anaerobically and streaked onto duplicate M17 plates. After another 2 days of
incubation one of the duplicate plates was used for screening. The colonies
were
transferred onto a nitrocellulose membrane and soaked for 1.5 minutes in
chloroform
for cell lysis. After cell lysis, the membrane was washed with distilled water
and dried
for 20 minutes. The membrane was subsequently incubated for 30 minutes in a
solution containing; 0.5 M Na-phosphate buffer (pH 6.1), 72 mM butanediol, 1
mM
NAD+ or NADP+, 0.02 mM Meldola's blue (8-Dimethylamino-2,3-benzophenoxazine),
0.08 mM MTT (3-[4,5-Dimethylthiazol-2yl) 2,5-diphenyltetrazolium bromide;
Thiazolyl
blue).


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(v) Protein electrophoresis

Native-PAGE was run at 150 V for 2.5 hours using 4-20% Tris-HCI gradient gels
with
Tris-Glycine (pH 8.3) as running buffer. Staining of native gels was performed
with the
zymogram technique (see below).

(vi) Zymogram staining of gels

Zymogram staining of native-PAGE gels for identification of diacetyl reductase
activity,
acetoin reductase activity and butanediol dehydrogenase activity was performed
as
follows: for diacetyl reductase activity the gel was incubated for 15 minutes
with 12
mM diacetyl, 1.5 mM NADH or NADPH, 0.5 M Na-phosphate buffer (pH 6.1), for
acetoin reductase activity the gel was incubated for 15 minutes with 36 mM
acetoin,
1.5 mM NADH or NADPH, 0.5 M Na-phosphate buffer (pH 6.1) and for butanediol
dehydrogenase activity the gel was incubated for 15 minutes with 72 mM
butanediol,
1 mM NAD+ or NADP+, 0.5 M Na-phosphate buffer. The gel was next incubated for
30 minutes under dry conditions before the addition of a solution consisting
of 0.02
mM Meldola's blue (8-Dimethylamino-2,3-benzophenoxazine), 0.08 mM MTT (3-(4,5-
Dimethylthiazol-2y1) 2,5-diphenyltetrazolium bromide; Thiazolyl blue) in 100
mM
phosphate buffer (pH 8.2) (Provecho et al, 1984; Gibson et al, 1991). Visible
protein
bands appeared within 20 minutes.

(vii) Cell-free extracts

DB1334, MW008 and MM084 were cultivated in M17 (0.5% glucose) until mid
exponential phase. The cells were harvested by centrifugation at 6000 rpm for
15
minutes and washed in cold 50 mM Na-phosphate buffer (pH 6). The pellet was
resuspended in cold 50 mM Na-phosphate buffer (pH 6) and sonicated for 3 x 2
minutes. The sonicated cell mixture was centrifuged at 6000 rpm for 15 minutes
and
the supernatant was stored at -20 C until analysed for protein concentration
and
enzyme activities.


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(vii) Enzyme activity measurements

Diacetyl reductase activity was measured spectrophotometrically by monitoring
the
oxidation of NADH or NADPH at 340 nm in a reaction mixture with the following
composition: 50 mM Na-phosphate buffer (pH 6.1), 36 mM diacetyl and 0.5 mM
NADH or NADPH. Butanediol dehydrogenase activity was measured spectrophoto-
metrically by monitoring the reduction of NAD+ or NADP ' at 340 nm in a
reaction
mixture with the following composition: 50 mM Na-phosphate buffer (pH 6.1), 72
mM
butanediol and 0.5 mM NAD+ or NADP+. Acetoin reductase activity was measured
spectrophotometrically by monitoring the oxidation of NADH or NADPH at 340 nm
in
a reaction mixture with the following composition: 50 mM Na-phosphate buffer
(pH
6.1), 36 mM acetoin and 0.5 mM NADH or NADPH. Lactate dehydrogenase activity
was measured by monitoring the oxidation of NADH at 340 nm in a reaction
mixture
with the following composition: 50 mM Tris-acetate buffer (pH 6), 0.5 mM
fructose-
1,6-diphosphate, 25 mM pyruvate and 0.5 mM NADH. The specific activities of
the
enzymes were expressed as micromoles of converted substrate per milligram of
protein per minute (equivalent to units per milligram protein).

(viii) Protein determination
Protein concentration was measured by using the BCA Protein Assay Reagent
(Pierce)
with bovine serum albumin as the standard.

2.3. Results
(i) Screening of EMS mutagenized MW008

Based on the previous results that diacetyl reductase is also able to react
with
butanediol and NADP+ (see Example 1), mutagenized MW008 was screened by
incubating the cells in a solution consisting of butanediol and NADP+.
Possible
mutants were colourless whereas cells with an intact diacetyl reductase were
stained
purple. Approximately 3500 clones were screened with the zymogram method. Two
possible mutants were further restreaked three times and repeatedly restained
with


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Meldola's blue and MTT. One of the two possible clones was found to be an
NADPH-
dependent diacetyl reductase mutant. This clone was designated MM084.

A sample of the mutant Leu. pseudomesenteroides MM084 strain was deposited in
accordance with the Budapest Treaty with the Deutsche Sammlung von
Mikroorganismen and Zellkulturen (DSMZ), Marschenroder Weg, 1b, D-38124
Braunschweig on 28 October 1998 under the Accession No. DSM 12465.

(ii) Enzyme activity measurements
Cell free extracts of DB1334, MW008 and MM084 were used for measuring diacetyl
reductase, acetoin reductase and butanediol dehydrogenase activities. As a
positive
control for the activity of the strains, lactate dehydrogenase activity was
also
measured. The enzyme activities of DB1334, MW008 and MM084 are summarised in
Table 2.1. Values for lactate dehydrogenase activities of the mutants were
comparable to the wild-type strain (data not shown).

Table 2.1. Diacetyl reductase (DR), acetoin reductase (AR), and butanediol
dehydrogenase (BUTDH) activities from cell free extracts of DB1334, MW008, and
MM084.

strain specific activity (U/mg)
DR AR BUTDH
NADH NADPH NADH NADPH NAD+ NADP+
DB1334 2.81 1.47 0.81 0.77 0.34 0.16
MW008 0.017 1.27 n.d 0.84 n.d 0.14
MM084 n.d n.d n.d n.d n.d n.d

n.d = not detectable, activities below the detection limit <0.005 U/mg.
(iii) Zymogram stained native-PAGE gels

Native-PAGE gels run with cell extracts from DB1334 and MM084 and incubated
with
diacetyl + NADPH, and butanediol + NADP+ showed that MM084 possessed no
activities with these substrates and cofactors (Fig. 2).


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2.4. Conclusions

It has been shown that wild-type Leu. pseudomesenteroides is capable of
reducing
diacetyl into acetoin and butanediol due to diacetyl reductase activities
using either
5 NADH or NADPH as cofactors.

An NADH-dependent diacetyl reductase mutant was capable of reducing diacetyl
at
the same rate as that of the wild-type strain during milk fermentation. When
using
NADPH as cofactor, the mutant had enzyme activities comparable to the wild-
type
10 strain. Mutagenesis and screening of MW008 with the zymogram technique
resulted
in the isolation of an NAD(P)H-dependent diacetyl reductase mutant. Such a
mutant
would be incapable of reducing diacetyl into acetoin and butanediol by means
of
diacetyl reductase.

EXAMPLE 3

Effect of diacetyl reductase deficient Leuconostoc pseudomesenteroides strain
MM084 on diacetyl stability in fermented dairy products under storage
3.1. Introduction

As described in Example 2, the diacetyl reductase mutant MM084 is isolated as
a
double mutant of Leuconostoc pseudomesenteroides strain DB1334 and lacks both
NADH and NADPH dependent DR.

When cultivated in milk as a pure culture, MM084 does not reduce diacetyl and
acetoin. Due to this characteristic, mutant MM084 is assumed to be a suitable
strain
for use as a component in mesophilic cultures which results in an improved
diacetyl
stability in the fermented products. In this Example, the effect of the mutant
MM084
on flavour formation and stability in fermented milk was investigated with
main focus
on the concentration of diacetyl.


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26

3.2. Materials and methods
(i) Bacterial strain

The strains used in this example originate from the Chr. Hansen Culture
Collection:
Lactococcus lactis subsp. lactis DB1 387 (0 strain);
Lactococcus lactis subsp. lactis biovar. diacetylactis strain DB1341 (D
strain);
Lactococcus lactis subsp. lactis biovar. diacetylactis -acetolactate
decarboxylase
deficient mutant MC010 (Curic et al. 1999) (D strain);
Leuconostoc pseudomesenteroides 13131334 (L strain);
Leuconostoc pseudomesenteroides DR- mutant MM084 (L strain).

Mixed cultures were composed of three different strains. Strains were produced
as
frozen pellets and stored at -50 C. The prepared inoculum contained 1.5 x 108
CFU/ml.

The following mixed culture were used in this example:
A)DB1387 + 13131341 + DB1 334
B) 13131387 + 13131341 + MM084
C) DB1387 + MC010 + DB1334
D) DB1387 + MC010 + MM084
Mixed culture A and C were used as a control.

iii) Cultivation medium

Reconstituted skimmed milk (9.5%); 200 ml in 250 ml bottles. Cream:
Commercially
available dairy cream (13%) was adjusted to 11 % fat by addition of skimmed
milk,
distributed in 500 ml bottles, re-pasteurised at 85 C for 30 min and cooled to
22 C
prior to inoculation.


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27

(iii) Cultivation conditions

The milk or cream was inoculated with a total of 1 % of inoculum and incubated
at
22 C until pH reached 4.60 0.05. Following incubation, the bottles were kept
at
4 C.

(iv) Determination of fermentation product formation

Samples for analysing the product formation were taken immediately after
inoculation,
during fermentation and during storage. Concentrations of the volatile
compounds
ethanol, acetaldehyde, -acetolactate, acetoin and diacetyl were determined by
HSGC
(Richelieu et al., 1997).

3.3 Results and discussion

No significant differences in the acidification rate were observed between
milk
fermented with cultures containing DR- MM084 (B and D) and the control mixed
cultures (A and C) (results not shown). At the end of the fermentation, all
four mixed
cultures produced similar amounts of ethanol and acetaldehyde. In all mixed
cultures
except culture D, acetaldehyde was reduced until the end of the fermentation
(results
not shown). Excess of acetaldehyde may cause yoghurt-like flavour of
buttermilk,
which is considered as an off-flavour. However, the concentration of
acetaldehyde
with mixed culture D is reduced during the first 2-3 days of storage.

It is shown that reduction of the diacetyl reductase activity of the
Leuconostoc strain
MM084 has a significant effect on the stability of diacetyl during storage
(Tables 3.1
and 3.2, Fig. 3 and 4). The diacetyl content in the medium at the end of the
fermentation is significantly higher in mixed cultures containing MM084 (B and
D) as
compared to the control mixed cultures (A and Q.
35


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28

Table 3.1. Diacetyl concentrations in milk, fermented by the mixed cultures A
and B,
during fermentation and storage.

Time (h) Diacetyl (mg/L)
Mixed culture A Mixed culture B
Fermentation 0 0.0 0.0
13 0.3 0.3
15 1.2 0.7
17 2.0 1.1
19 1.8 1.4
21 1.0 1.6
22 0.5 1.4
Storage 46 (+ 1 day) 0.3 0.9
94 (+ 3days) 0.2 0.8
142 (+ 5days) 0.0 0.7
190 (+ 7days) 0.0 0.6
358 (+ 14days) 0.0 0.6
502 (+ 20days) 0.0 0.7

Table 3.2 Diacetyl concentrations in sour cream fermented by the mixed
cultures C
and D during fermentation and storage.

Time (h) Diacetyl (mg/L)
Mixed culture C Mixed culture D
Fermentation 0 0.0 0.0
12 3.7 2.3
13 5.5 4.6
14 7.1 9.7
9.4 12.8
16 8.5 12.4
17 8.0 9.6
Storage 41 (+ 1 day) 1.9 5.4
113 (+ 4days) 0.5 4.6
161 (+6days) 0.6 4.2
305 (+ 12days) 0.3 2.0
689 (+ 28days) 0.1 0.8
857 (+ 35days) 0.1 0.9
1025 (+42 days) 0.1 1.0


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The sour cream fermented with the cultures C and D was sensorically evaluated
after
1, 7, 14, 21, and 28 days, respectively. The sour cream had a mild, clean and
fresh
flavour. The fresh flavour was maintained during a prolonged storage.

3.4. Conclusion

The strain MM084 is suitable for use as a component of a mixed aroma-forming
culture. The mixed cultures composed with MM084 had a significantly improved
diacetyl stability during storage and a significant higher content of diacetyl
at the end
of fermentation and after storage. Such a mixed culture is beneficial in the
production
of sour cream and cream cheeses.

EXAMPLE 4
Isolation and characterisation of a Lactococcus /actis subsp. lactis mutant
with
enhanced diacetyl reductase activity

4.1. Introduction
The L. lactis subsp. lactis mutant strain DN223 is both a lactate
dehydrogenase (LDH)
and pyruvate formate lyase (PFL)defective. DN223 is strictly aerobic and the
lack of
capability to grow anaerobically (even in the presence of acetate) is most
likely due to
a constraint on the intracellular redox balance, as the net consumption of
NAD+ in the
glycolysis can no longer be regenerated due to the two enzymatic defects.
Exogenous
acetoin was expected to assist in the regeneration of NAD+ under anaerobic
conditions by conversion into 2,3-butanediol by the enzyme diacetyl reductase
(DR).
4.2. Isolation of a mutant with enhanced DR activity

A test tube containing 10 ml of DN medium (Dickely et al., 1995) supplemented
with
acetate was inoculated with a single colony of DN223 picked from an agar plate
and
incubated aerobically overnight at 30 C. 100 l of the overnight culture was
spread
onto two agar plates containing DN medium supplemented with 2.0 g/L sodium


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acetate trihydrate and 0.5 g/L acetoin and incubated anaerobically for two
days at
30 C. A number of colonies were subsequently streaked onto agar plates
containing
DN medium supplemented with acetate and with and without 0.5 g/L acetoin and
incubated anaerobically for two days at 30 C. One mutant designated CMH-1 53
was
5 isolated which only displayed anaerobic growth if the medium was
supplemented with
0.5 g/L acetoin.

A sample of the L. lactis subsp. lactis CMH-1 53 strain was deposited in
accordance
with the Budapest Treaty with the Deutsche Sammlung vor Mikroorganismen and
10 Zellkulturen (DSMZ), Marschenroder Weg, 1 b, D-38124 Braunschweig on 7
April
1998 under the Accession No. DSM 12096.
4.3. Characterisation of L. lactis CMH-1 53

15 200 ml of DN medium supplemented with acetate was inoculated with a single
colony
of CMH-1 53 picked from an agar plate and incubated aerobically overnight at
30 C.
Subsequently, a cell-free extract was made and the protein content of the
extract was
measured.

20 The diacetyl activities of strain CMH-1 53 were measured with diacetyl as
substrate
and NADH as cofactor and are expressed as the units of [ moles NADH consumed
per
min. per mg of protein] according to the assay described in Example 1. The
diacetyl
activities of strain CMH-1 53 were compared with other L. lactis subsp. lactis
strains
(Table 4.1).
Additionally, the diacetyl activities of strain CHM-153 were measured using
either
diacetyl, acetoin or 2,3-butanediol as substrate and NADH, NAD+, NADPH or
NADP+
as cofactor and are expressed as units of [ moles NADH or NADPH produced or
consumed per min. per mg of protein] according to the assay described in
Example 1.
The diacetyl activities of strain CMH-1 53 were compared with other L. lactis
subsp.
lactis strains (Table 4.2).


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31

Table 4.1. Diacetyl reductase activity of CMH-153 compared with other L.
lactis
subsp. lactis strains.

Strain Phenotype Specific activity
CHCC373 Wild-type n.d.
DN221 PfI- n.d.
DN223 Pfl"/Ldh" 0.01-0.02
DN224 Ldh- 0.01
CMH-153 Pff/Ldh'/Dr++ 0.92

n.d = not detectable, activities below the detection limit <0.005 U/mg.

Table 4.2 Diacetyl reductase activity of CMH-1 53 compared with other L.
lactis
subsp. lactis strains

Strains
Enzymatic reaction Co-factor CHCC373 DN223 CMH-153
diacetyl ---> acetoin NADH n.d. n.d. 1.15
NADPH n.d. n.d. 0.04
acetoin ---> diacetyl NAD+ n.d. n.d. n.d.
NADP+ n.d. n.d. n.d.
acetoin ---> 2,3-butanediol NADH 0.02 0.07 0.41
NADPH n.d. n.d. 0.02
2,3-butanediol ---> acetoin NAD+ n.d. n.d. 0.12
F7 NADP+ n.d. n.d. n.d.
n.d = not detectable, activities below the detection limit <0.005 U/mg.

Finally, the specific LDH activity of strain CMH-1 53 was measured using the
method
as also described in Example 1 and compared with other L. lactis subsp. lactis
strains
(Table 4.3). Activities are expressed as units of [ moles NADH consumed per
min. per
mg of protein].



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32

Table 4.3. Lactate dehydrogenase activity of CMH-1 53 compared with other L.
lactis
subsp. lactis strains

Strain Phenotype Specific activity
CHCC373 Wild-type 15.3
DN221 Pfl' 15.3
DN223 Pfl'/Ldh- n.d.
DN224 Ldh" n.d.
CMH-153 Pfl'/Ldh'/Dr++ n.d.

n.d = not detectable, activities below the detection limit <0.005 U/mg.

The specific diacetyl reductase activities of CMH-1 53 are significantly
increased
compared to other L. lactis strains with various phenotypes (Table 4.1 and
4.2)
whereas CMH-1 53 has no detectable LDH activity (Table 4.3). Thus, the mutant
strain
L. lactis subsp. lactis CMH-1 53 has the phenotype Ldh'/Pfl-/Dr++, as it is
only capable
of anaerobic growth if supplied with acetoin and acetate.


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33

REFERENCES
Arora, B.C., Dutta, S.M., Sabharwal, V.B. and Ranganathan, B. (1978). Mutants
of
Streptococcus lactis subsp. diacetylactis lacking diacetyl reductase activity.
Acta
Microbiol Pol 27:353-358.

Boumerdassi, H., Monnet, C., Desmazeaud and M., Corrieu, G. (1997). Isolation
and
properties of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483
mutants
producing diacetyl and acetoin from glucose. Appl. Environ. Microbiol. 63:
2293-
2299.

Crow, V.L. (1990). Properties of 2,3-butanediol dehydrogenases from
Lactococcus
lactis subsp. lactis in relation to citrate fermentation. Appl. Environ.
Microbiol.
56:1656-1665.
Curic, M., Lauridsen, B. S., Renault, P. and Nilsson, D. (1999). A general
method for
selection of -acetolactate decarboxylase deficient Lactococcus lactis mutants
to
improve diacetyl formation. Appl. Environ. Microbiol. 63:1202-1206.

Dickely, F., Nilsson, D., Hansen, E.B. and Johansen, E. (1995). Isolation of
Lactococcus lactis nonsense suppressors and construction of a food-grade
cloning
vector. Mol. Microbiol. 15:839-847.

Gibson, T.D., Parker, S.M. and Woodward, J.R. (1991). Purification and
characterization of diacetyl reductase from chicken liver and Streptococcus
lactis and
enzymatic determination of diacetyl and ketones. Enz. Microb. Technol. 13:171-
178.
Giovannini, P.P., Medici, A., Bergamini, C.M. and Rippa, M. (1996). Properties
of
diacetyl (acetoin) reductase from Bacillus stearothermophilus. Bioorg. Med.
Chem.
4:1197-1201.

Kulia, R.K. and Ranganathan, B. (1978). Ultraviolet light-induced mutants of
Streptococcus lactis subsp. diacetylactis with enhanced acid- or flavor-
producing
abilities. J. Dairy Sci. 61:379-383.


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34

Provecho, F., Burgos, J. and Sarmiento, R.M. (1984). Further purification and
characterization of diacetyl reducing enzymes from beef liver. Int. J.
Biochem.
16:423-427.
Richelieu, M., Houlberg, U. and Nielsen, J.C. (1997). Determination of a-
acetolactic
acid and volatile compounds by headspace gas chromatography. J. Dairy Sci.
80:1918-1925.

Terzaghi, B.E. and Sandine, W.E. (1975). Improved medium for Lactic
streptococci
and their bacteriophages. Appl. Microbiol. 29:807-813.


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INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13bis)

A. The indications made below relate to the microorganism referred to in the
description
on page 30 line 8

B. IDENT7FICATTON OF DEPOSIT Further deposits are identified on an additional
sheet
Name of depositary institution

DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH
Address of depositary institution (hidading postal code and country)
Mascheroder Weg 1B
D-38124 Braunschweig
Germany

Date of deposit Accession Number
7 April 1998 DSM 12096

C. ADDITIONAL INDICATIONS (le,.veblank if not applicable) This information is
continued on an additional sheet 0
As regards the respective Patent offices of the respective desig-
nated states, the applicants request that a sample of the deposi-
ted microorganisms only be made available to an expert nominated
by the requester until the date on which the patent is granted or
the date on which the application has been refused or withdrawn or
is deemed to be withdrawn.
D. DESIGNATED STATES FOR-WMCH INDICATIONS ARE MADE (iftlra inlaaoioar ere not
for alllaiprweyiS Ater)

E. SEPARATE FURNISmNG OIKINDICATIONS (len'c Weak ijaot applicable)
The indiationslistedbelowwili besubaitted to the International Bureau later
(spoaijyL4cgo ereinwareojr4cindmtiowcg, 'Aaeoioe
Numberof Depoirit9

For receiving Office use only For International Bureau use only
This sheet was received with the international application [J This sheet was
received by the International Bureau on:
Authorized officer Authorized officer

Frain P( tlRnn'" I..w IW,-%


CA 02326405 2000-10-19

WO 99/54453 PCT/DK99/00218
36

INDICATIONS RELATING TO DEPOSITED MICROORGANISMS
(PCT Rule 12bis)

Additional sheet

In addition to the microorganism indicated on page 35 of the description, the
following
microorganisms have been deposited with

DSM-Deutsche Sammlung von Mikroorganismen and Cellkulturen GmbH
Mascheroder Weg 1 b, D-381 24 Braunschweig, Germany

on the dates and under the accession numbers as stated below:
Accession Date of Description Description
number deposit Page No. Line No.
DSM 12099 7 April 1998 19 4
DSM 12465 28 Oct.1998 24 4

For all of the above-identified deposited microorganisms, the following
additional
indications apply:

As regards the respective Patent Offices of the respective designated states,
the
applicants request that a sample of the deposited microorganisms stated above
only
be made available to an expert nominated by the requester until the date on
which the
patent is granted or the date on which the application has been refused or
withdrawn
or is deemed to be withdrawn.

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Forecasted Issue Date 2011-01-04
(86) PCT Filing Date 1999-04-20
(87) PCT Publication Date 1999-10-28
(85) National Entry 2000-10-19
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