Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1999-04-21
2002-07-02
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S189000, C435S190000, C435S252100, C435S440000, C435S441000, C435S448000
Reexamination Certificate
active
06413765
ABSTRACT:
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 modified 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 latcis
are among the most commonly used lactic acid bacteria in dairy starter cultures. Several other lactic acid bacteria such as Leuconostoc species, Pidococcus species. Lactobacillus species, Oenococcus species and Streptococcus species are also commonly used in food 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, &agr;-acetolactate, acetoin, diacetyl, carbon dioxide and 2,3 butylene glycol (butanediol).
Among these metabolites, diecetyl (2,3-butanedione) is an essential flavour compound in dairy products such as butter, yoghurt, starter distilate, 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, Leuconostc and Lactobacillus by an oxidative decarboxylation of &agr;-acetolactate which is formed from two molecules of pyruvate by the action of &agr;-acetolactate synthase (ALS)
Diacetyl reducing enzymes, commonly termed diacetyl reducteses (DR) (acetoin:NAD oxidoreductases E. C. 1.1.1.5), have been observed from many different sources, notably animal tissues (Provecho et al., 1984), bacteria including Lactococcus (formerly
Streptococcus
)
lactis
(Crow, 1990; Arora et al., 1978), Bacillus species and Enterbacter species (Giovannini et al. 1996), and yeast (Gibson et al., 1991). Boumerdassi et al. 1997 disclosed a mutated
Lactococcus latis
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 & Rangenathan 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 “discetyl reductase” (“DR”) encompasses several enzymatic activities such as diacetyl reductase activity, acetoin reductese 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 acetoin and/or butanediol, a characteristic that is generally undesirable in the production of dairy products. The enzyme responsible for the reduction of diacetyl, diacetyl reductase, is highly expressed in Leuconostoc species such as
Leu. psoudomesenteriodes
which species is known to have about 100 times higher diacetyl reductese activity than
L. lactis.
Thus, one primary objective of the present invention is to provide lactic acid bacterial cultures of species, including Leuconastoc 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
(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
(vi) 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 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 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
Henriksen Claus Maxel
Nilsson Dan
Walfridsson Mats
Chr. Hanson A/S
Foley & Lardner
Fronda Christian L.
Nashed Nashaat T.
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