Compositions prepared to contain active dextran sucrase...

Details Compositions prepared to contain active dextran sucrase... Compositions prepared to contain active dextran sucrase...

1999-11-12

2001-05-22

Prats, Francisco (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S200000, C435S211000, C435S101000, C435S103000, C536S112000, C424S094610, C426S056000

Reexamination Certificate

active

06235511

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to dextran sucrase produced by bacteria and means for production of the same and its use.
STATE OF THE ART
Dextran is a polysaccharide formed of glucose units, the chain lengthening of which is catalyzed by dextran sucrase. The biosynthesis of dextran has been demonstrated in numerous bacteria, especially in
Streptococcus mutans, Leuconostoc mesenteroides
ssp.
mesenteroides
and
Leuconostoc mesenteroides
ssp.
dextranicum.
Leuconostoc produce the enzyme dextran sucrase and secrete it into the culture medium in the presence of sucrose. This enzyme, dextran sucrase, then synthesizes dextran from the sucrose substrate. Dextran has applications in several fields. It is used especially in biochemistry as a support for filtration chromatography on a gel of the Sephadex type. Additionally, in the field of therapeutics, it is used as a substitute for blood plasma (Biochimie générale (General Biochemistry)—J. H. WEIL—Masson, 6th edition—1990—p. 171).
Furthermore, dextran synthesized by a strain of
Leuconostoc dextranicum
is applied in the food industry for the texturing of food products such as yoghurts, cream desserts, milk-based drinks and salad dressings. European Patent Application Publication No. EP 0 363 633 demonstrates the synthesis of dextran by a strain of
Leuconostoc dextranicum
and in particular by the strain
Leuconostoc dextranicum
NRRL-B-18242. Additionally , that patent application publication describes especially a composition containing dextran synthesized by this bacterium and the use of this composition in the food sector.
Moreover, the taxonomy of the bacterial strains of the genus Leuconostoc has been revised several times.
Garvie et al. (International Journal of Systematic Bacteriology, 118-119, 1983) describe the taxonomy of the bacterial strains of the genus Leuconostoc, established according to a criterion of homology in terms of the deoxyribonucleic acid (DNA). The bacteria, previously classed as
Leuconostoc mesenteroides, Leuconostoc dextranicum
and
Leuconostoc cremoris,
although of different phenotype, have a very high degree of homology in respect of their DNA. Therefore, according to this taxonomy, these bacteria are subspecies of
Leuconostoc mesenteroides
and are respectively called
Leuconostoc mesenteroides
ssp.
mesenteroides, Leuconostoc mesenteroides
ssp.
dextranicum
and
Leuconostoc mesenteroides
ssp.
cremoris.
In addition, J. B. Milliere et al. (Journal of Applied Bacteriology, 67, 529-542, 1989) describe a taxonomic analysis performed on 81 strains of the genus Leuconostoc, including 11 strains of
Leuconostoc mesenteroides
ssp.
cremoris.
This analysis relies on the taxonomy established by Garvie et al. and is based on the following criteria in particular: the capacity of these strains to ferment various sugars, the capacity of these strains to utilize citrate and the capacity of these strains to produce dextran. This document notes the fact that a strain of
Leuconostoc mesenteroides
ssp.
cremoris
does not synthesize dextran. Also, such a strain is distinguished and defined by the fact that it does not ferment pentoses.
No strain of
Leuconostoc mesenteroides
ssp.
cremoris
has yet been isolated which is capable of synthesizing dextran. Now,
Leuconostoc mesenteroides
ssp.
cremoris
is of major importance in the manufacture of dairy products such as, for example, yoghurt-type fermented specialities or dairy creams. It would therefore be very valuable to be able to use such bacteria, capable of synthesizing dextran of pleasant texture and taste, especially for texturing this type of food product.
The object of the present invention is to meet this need.
SUMMARY OF THE INVENTION
For meeting the need noted above the present invention provides strains of
Leuconostoc mesenteroides
ssp.
cremoris
which produce dextran, especially the strains
Leuconostoc mesenteroides
ssp.
cremoris
CNCM I-1692 and CNCM I-1693.
The present invention further provides a method of using a strain of
Leuconostoc mesenteroides
ssp.
cremoris
for the manufacture of a food product or cosmetic composition.
The present invention further provides a process for the production of dextran from
Leuconostoc mesenteroides
ssp.
cremoris.
The present invention further provides a food composition or cosmetic composition into which dextran obtained by carrying out the process is incorporated during its preparation.
The present invention further provides a process for the production of an additive containing active dextran sucrase from
Leuconostoc mesenteroides
ssp.
cremoris.
The present invention finally relates to a food product or cosmetic composition which contains active dextran sucrase produced by
Leuconostoc mesenteroides
ssp. including a composition into which an additive containing active dextran sucrase from
Leuconostoc mesenteroides
ssp.
cremoris
is incorporated during its preparation.
DETAILED DESCRIPTION OF THE INVENTION
As described above, present invention provides strains of
Leuconostoc mesenteroides
ssp.
cremoris
which produce dextran. It was possible to isolate strains of
Leuconostoc mesenteroides
ssp.
cremoris
which produce dextran. All the strains of
Leuconostoc mesenteroides
ssp.
cremoris
which produce dextran are therefore covered by the present invention.
A strain of
Leuconostoc mesenteroides
ssp.
cremoris
was isolated particularly from a Swiss cream and it was found, surprisingly, to have the remarkable property of synthesizing dextran of pleasant texture and taste. This strain was deposited on Apr. 18, 1996, under the terms of the Budapest Treaty, in the Collection National de Cultures de Microorganismes, INSTITUT PASTEUR, 25, rue du Docteur Roux, F-75724 PARIS CEDEX 15, where it was given the deposit number CNCM I-1692.
Furthermore, a strain of
Leuconostoc mesenteroides
ssp.
cremoris
which also has the remarkable property of synthesizing dextran of pleasant texture and taste was isolated by natural selection from the strain CNCM I-1692. This strain was deposited on Apr. 4, 1996, under the terms of the Budapest Treaty, in the Collection Nationale de Cultures de Microorganismes, INSTITUT PASTEUR, 25, rue du Docteur Roux, F-75724 PARIS CEDEX 15, where it was given the deposit number CNCM I-1693.
Details of these strains, concerning especially their morphology, the fermentation of sugars and other aspects, are given below.
Morphology
Gram-positive microorganisms,
Negative catalase,
Facultative aerobe, and
Cocci.
Fermentation of Sugars
No lactic acid production from pentoses, D- and L-arabinose, D- and L-xylose and D- and L-ribose,
Lactic acid production from lactose by the strain CNCM I-1692, and
No lactic acid production from lactose by the strain CNCM I-1693.
Other Aspects
Strains synthesizing dextran, a polysaccharide with remarkable texturing properties.
According to the present invention, it is therefore possible also to isolate a strain of
Leuconostoc mesenteroides
ssp.
cremoris
which does not ferment lactose, an example being CNCM I-1693.
A preferred strain according to the present invention produces the same dextran as the strain CNCM I-1692 or the strain CNCM I-1693.
As indicated above, present invention provides a process for the production of dextran wherein a medium containing sucrose is inoculated with a preculture of a strain of
Leuconostoc mesenteroides
ssp.
cremoris
according to the invention, it is allowed to ferment at 25-35° C. for 10-20 h and the pH of the resulting culture is then lowered to 5-5.5 prior to storage at 0-10° C. for 16-48 h.
A medium containing at least 2% of sucrose can be inoculated with a preculture of a strain of
Leuconostoc mesenteroides
ssp.
cremoris
according to the invention, for example in order to allow the production of dextran sucrase and the synthesis of dextran in the culture medium.
A medium containing 5-12% of MSK medium (skimmed cow's milk) supplemented with 0.05-0.2% of yeast extract and at least 2% of sucrose, for example, can be inoculated with 0.2-3% of a precultu

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