Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2001-06-13
2003-12-09
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C426S034000, C435S041000, C435S139000, C435S170000, C435S243000, C435S252900
Reexamination Certificate
active
06660515
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of lactic acid bacterial starter cultures and in particular there is provided the means of enhancing the growth rate and/or controlling the metabolic activity of lactic acid bacteria by cultivating the lactic acid bacteria in association with a lactic acid bacterial helper organism which has a defeat in its pyruvate metabolism. Such a helper organism is also useful as a means of improving the shelf life and/or quality of edible products.
TECHNICAL BACKGROUND AND PRIOR ART
Lactic acid bacteria are used extensively as starter cultures in the food industry in the manufacture of fermented products including milk products such as e.g. yoghurt and cheese, meat products, bakery products, wine and vegetable products.
As used herein the term “lactic acid bacteria” refers to gram-positive, microaerophilic or anaerobic bacteria which ferment sugars 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, such as
Lactococcus lactis,
Lactobacillus species, Streptococcus species, Leuconostoc species, Pediococcus species and Propionibacterium species. Also the strict anaerobes belonging to the genus Bifidobacterium is generally included in the group of lactic acid bacteria.
When a lactic acid bacterial starter culture is added to milk or any other edible starting material and appropriate conditions for growth and metabolic activity of the starter culture are provided, the bacteria will start to propagate after a period of time known as the lag phase, during which the bacteria adapt to the new conditions. Once propagation of the bacteria is initiated it is rapid with concomitant conversion of citrate, lactose or other sugars into lactic acid/lactate as the major acidic metabolite, and possibly other acids including acetate, resulting in a pH decrease. In addition, several other metabolites such as e.g. acetaldelyde, &agr;-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol) are produced during the growth of the lactic acid bacteria.
Generally, the growth rate and the metabolic activity of lactic acid bacterial starter cultures can be controlled by selecting appropriate growth conditions for the strains of the specific starter culture used such as appropriate growth temperature, oxygen tension and content of nutrients. Thus, it is known in the dairy industry that a reduction of the oxygen content of the milk raw material will result in a more rapid growth of the added lactic acid bacteria which in turn results in a more rapid acidification of the inoculated milk. Currently, such a reduction of the oxygen content is carried out by heating the milk in open systems, by deaerating the milk in vacuum or by a sparging treatment. Alternative means of reducing the oxygen content include the addition of oxygen scavenging compounds.
Lactic acid bacterial starter cultures are commonly used in the food industry as mixed strain cultures comprising one or several species. For a number of mixed strain cultures such as yoghurt starter cultures typically comprising strains of
Lactobacillus bulgaricus
and
Streptococcus thermophilus,
a symbiotic relationship between the species has been reported, assumingly due to proteolytic activity of at least one of the strains (Rajagopal et al. J. Dairy Sci., 1990 73:894-899). It has also been reported that in such mixed yoghurt cultures, stimulation of growth of the Lactobacillus component is due to the inherent formation of formic acid by the
Streptococcus thermophilus
(Suzuki et al., 1986). A further example of a symbiotic relationship between strains in a mixed culture is disclosed in EP 0 111 392 A2 where it is demonstrated that selected wild-type
Streptococcus thermophilus
strains having a relatively high oxygen uptake ability improves the survival of a strictly anaerobic Bifidobacterium species when it is combined with the Streptococcus strain.
However, the prior art is not aware of any generally applicable biological method whereby the growth and metabolic activity of lactic acid bacterial starter cultures can be enhanced. It is therefore a primary objective of the present invention to provide such a method.
SUMMARY OF THE INVENTION
Accordingly, the present invention relates in a first aspect to a method of enhancing the growth rate and/or controlling the metabolic activity of a lactic acid bacterial strain, comprising cultivating the strain in association with a lactic acid bacterial helper organism that is defective in its pyruvate metabolism.
In a further aspect there is provided a method of improving the shelf life and/or the quality of an edible product comprising adding to the product a lactic acid bacterial strain that is defective in its pyruvate metabolism and in a still further aspect the invention pertains to a starter culture composition comprising a lactic acid bacterium and a lactic acid bacterial helper organism that in defective in its pyruvate metabolism, said helper organism being capable of enhancing the growth rate and/or controlling the metabolic activity of the lactic acid bacterium.
In yet another aspect the invention pertains to a lactic acid bacterium that is defective in at least one enzyme involved in the pyruvate metabolism and in which a gene capable of regenerating NAD
+
is overexpressed.
DETAILED DISCLOSURE OF THE INVENTION
It is a primary objective of the present invention to provide a generally applicable method of enhancing the growth rate and/or controlling the metabolic activity of a lactic acid bacterial starter culture. The method comprises cultivating the culture in association with a lactic acid bacterial helper organism which is defective in its pyruvate metabolism.
It will be understood that enhancement of the growth rate relates to any effect resulting in a higher number of starter culture cells in the medium after a given period of time, i.e. the lactic acid bacterial cells propagate at a higher rate than that obtained without the helper organism, or the cells start propagating at an earlier point in time at a rate equal to or higher as compared to propagation of the lactic acid bacteria without the helper organism.
As used herein, the expression “controlling the metabolic activity” refers to the increased or decreased production of any metabolite produced by the starter culture, including the production of acids, such as lactic acid, acetic acid, formic acid and/or propionic acid. Examples of other metabolites of relevance, the production of which may be controlled, include aroma compounds such as acetaldehyde, &agr;-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol).
In accordance with the invention, the lactic acid bacterial helper organism is defective in its pyruvate metabolism. As used herein the expression “defective in its pyruvate metabolism” indicates that the helper organism in comparison with the parent strain has an altered metabolism of pyruvate, i.e. an increased or decreased production of one or more metabolites derived from pyruvate.
Such an altered metabolism of pyruvate can be the result of the helper organism being defective in its ability to egress at least one enzyme selected from the group consisting of pyruvate formate lyase, pyruvate dehydrogenase, lactate dehydrogenase, acetolactate synthetase, second acetolactate synthetase, acetolactate decarboxylase and diacetyl reductase. As used herein the expression “defective in its ability to express any of the above enzymes”, indicates that the helper organism as compared to the parent strain from which it is derived has a reduced production of the enzyme or that the enzyme is not expressed at all, irrespective of the growth conditions.
Examples of lactic acid bacterial helper organisms which are defective in their ability to express at least one of the above mentioned enzymes include the
Lactococcus lactis
subspecies
lactis
strain DN223 which is defective both in the pyruvate form
Kringelum Boerge
Nillson Dan
Soerensen Kim Ib
CHR. Hansen A/S
Cooper Iver P.
Lankford , Jr. Leon B.
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