Dextran production

Abstract

Dextran-producing strains of Leuconostoc mesenteroides ssp. cremoris, i.e. those registered at the CNCM under the designations l-1692 and l-1693, are new. Also claimed are: (a) production of dextran by fermenting a culture medium containing sucrose by inoculation with a preculture of a selected strain of Leuconostoc mesenteroides ssp. cremoris; and (b) recovering an additive containing the enzyme dextran-sucrase from the fermentation product.

Description

taining an enzyme involved in the biosynthesis of dextran, and to theof this dextran, this enzyme and / or a strain producing this dextran ands enzyme in 10 the manufacture of a food product or cosmeticposition. STATE OF THE ART Dextran is a polysaccharide formed of glucosets, the 15 chain lengthening of which is catalyzed by dextran sucrase.biosynthesis of dextran has been demonstrated in numerous bacteria,ecially in Streptococcus mutans, Leuconostoc mesenteroides ssp.enteroides and Leuconostoc mesenteroides ssp. 20 dextranicum.conostoc produce the enzyme dextran sucrase and secrete it into theture medium in the presence of sucrose. This enzyme, dextran sucrase,n synthesizes dextran from the sucrose substrate. Dextran haslications in several fields. It is used especially 25 in biochemistry asupport for filtration chromatography on a gel of the Sephadex type.itionally, in the field of therapeutics, it is used as a substitute forod plasma (Biochimie generate (General Biochemistry) - J.H. WEIL -son, 6th edition - Z990 - p. 171). 30 Furthermore, dextran synthesizeda strain of Leuconostoc dextranicum is applied in the food industry fortexturing of food products such as yoghurts, cream desserts, milk-basednks and salad dressings. Thus 35 EP 0363633 demonstrates the synthesisdextran by a strain of Leuconostoc dextranicum and in particular by theain Leuconostoc dextranicum NRRL-B-18242. Said document describesecially a composition containing dextran synthesized by this bacteriumthe use of this 40 composition in the food sector. 2 5 APR 2002 2eover, the taxonomy of the bacterial strains of the genus Leuconostocbeen revised several times. Thus Garvie et al. (International JournalSystematic 5 Bacteriology, 118-119, 1983) describe the taxonomy of theterial strains of the genus Leuconostoc, established according to aterion of homology in terms of the deoxyribonucleic acid (DNA). Theteria, previously classed as Leuconostoc mesenteroides, Leuconostoc 10tranicum and Leuconostoc cremoris, although of different phenotype, haveery high degree of homology in respect of their DNA. Therefore,ording to this taxonomy, these bacteria are subspecies of Leuconostocenteroides and are respectively called Leuconostoc 15 mesenteroides ssp.enteroides, Leuconostoc mesenteroides ssp. dextranicum and Leuconostocenteroides ssp. cremoris.moris. This analysis relies on the taxonomy established by Garvie et al.is based on the following 25 criteria in particular: the capacity ofse strains to ferment various sugars, the capacity of these strains tolize citrate and the capacity of these strains to produce dextran. Saidument notes the fact that a strain of Leuconostoc mesenteroides ssp.moris does not 30 synthesize dextran. Also, such a strain istinguished and defined by the fact that it does not ferment pentoses. Noain of Leuconostoc mesenteroides ssp. cremoris has yet been isolatedch is capable of synthesizing 35 dextran. Now, Leuconostoc mesenteroides. cremoris is of major importance in the manufacture of dairy productsh as, for example, yoghurt-type fermented specialities or dairy creams.would therefore be very valuable to be able to use such bacteria,able of synthesizing 40 dextran of pleasant texture and taste,ecially for texturing this type of food product. 3 2 5 APR 2002 Theect of the present invention is to meet this need. 5 For this purposepresent invention relates to a strain of Leuconostoc mesenteroides ssp.moris which produces dextran, especially the strains Leuconostocenteroides ssp. cremoris CNCM I-1692 and CNCM I-1693. 10 The presentention further relates to a method of using a strain of Leuconostocenteroides ssp. cremoris for the manufacture of a food product ormetic composition. 15 The present invention further relates to a processthe production of dextran from Leuconostoc mesenteroides ssp. cremoris.present invention further relates to a food product or 20 cosmeticposition into which dextran obtained by carrying out this process isorporated during its preparation. The present invention further relatesa process for the 25 production of an additive containing active dextranrase from Leuconostoc mesenteroides ssp. cremoris. The present inventionally relates to a food product or cosmetic composition into which anitive containing 30 active dextran sucrase from Leuconostocenteroides ssp. cremoris is incorporated during its preparation.AILED DESCRIPITI:ON OF THE INVENTION 35 The present invention thereforeates to a strain of Leuconostoc mesenteroides ssp. cremoris whichduces dextran. It was possible to isolate strains of Leuconostocenteroides ssp. cremoris which produce dextran. A11 the strains ofconostoc mesenteroidesprisingly, to have the remarkable property of 5 synthesizing dextran ofasant texture and taste. This strain was deposited on 18 / 04 / 96, underterms of the Budapest Treaty, in the Collection Nationale de CulturesMicroorganismes, INSTITUT PASTEUR, 25, rue du Docteur Roux, F-75724IS CEDEX 15, where it was given the 10 deposit number CNCM I-1692.thermore, a strain of Leuconostoc mesenteroides ssp. cremoris which alsothe remarkable property of synthesizing dextran of pleasant texture andte was 15 isolated by natural selection from the strain CNCM I-1692.s strain was deposited on 1S / 04 / 96, under the terms of the Budapestaty, in the Collection Nationale de Cultures de Microorganismes,TITUT PASTEUR, 25, rue du Docteur Roux, F-75724 PARIS CEDEX 15, where itgiven 20 the deposit number CNCM I-1693. Details of these strains,cerning especially their morphology, the fermentation of sugars ander aspects, are given below. 25 Morphology - Gram-positiveroorganisms, - Negative catalase, - Facultative aerobe, 30 - Cocci.mentation of sugars - No lactic acid production from pentoses, D- andabinose, D- and L-xylose and D- and L-ribose, 35 - Lactic acidduction from lactose by the strain CNCM I-1692, - No lactic acidduction from lactose by the strain CNCM I-1693. 2 5 APR 2002 5 Otherectss not ferment lactose, an example being CNCM I-1693. 10 A preferredain according to the present invention produces the same dextran as theain CNCM I-1692 or the strain CNCM I-1693. The present invention furtherates to a process for the 15 production of dextran wherein a mediumtaining sucrose is inoculated with a preculture of a strain ofconostoc mesenteroides ssp. cremoris according to the invention, it isowed to ferment at 25-35C for 10-20 h and the pH of the resultingture is then lowered to 5-5.5 prior to 20 storage at 0-10C for 16-48 h.edium containing at least 2% of sucrose can be inoculated with aculture of a strain of Leuconostoc mesenteroides ssp. cremoris accordingthe invention, 25 for example in order to allow the production oftran sucrase and the synthesis of dextran in the culture medium. Aium containing 5-12% of MSK medium (skimmed cow's 30 milk) supplementedh 0.05-0.2% of yeast extract and at least 2% of sucrose, for example,be inoculated with 0.2-3% of a preculture of a strain of Leucoaostocenteroides ssp. cremoris according to the present invention,ticularly the strain CNCM I-1692 or the 35 strain CNCM I-1693. Theium can be allowed to ferment at 25-35C for 10-20 h with the pH beingntained at 67.3, for example. Then, when fermentation has ended, the pHthe resulting culture can be lowered to 5-5.5 by the addition of lacticd, for example. The culture is then 40 stored at 0-10C for 16-48 h. 2PR 2002 6 Also, a medium containing 0.05-0.2% of yeast extract with atst 2% of sucrose, for example, can be inoculated with 0.2-3% of aculture of a strain of Leuconostoc mesenteroides ssp. cremoris accordingthe present 5 invention, particularly the strain CNCM I-1692 or theain CNCM I-1693. The medium can be allowed to ferment at 25-35C for20 h with the pH being maintained at 67.3, for example. Whenmentation has ended, this culture can be mixed with an equal volume ofMSK 10 medium so that inhibition of the production of the enzymetran sucrase by the lactose contained in the MSK medium, for example, isided during the fermentation. The pH of the resulting culture can thenlowered to 5-5.5 by the addition of lactic acid, for example. The 15ture is then stored at 0-10C for 16-48 h. This culture can then be driedgive a dextran powder, for example. This culture can be dried byphilization or spray drying, for example. 20 The present inventionther relates to a food product or cosmetic composition comprisingtran from Leuconostoc mesenteroides ssp. cremoris which is obtainable byrying out the above process. 25 To prepare such a product orposition, dextran obtained in this way can be incorporated into a foodcosmetic product, such as a milk powder, a yoghurt, a ketchup, aonnaise or a skin cream, during its manufacture, for 30 example.enteroides ssp. cremoris according to the invention and is then allowedferment at 25-35C for 10-20 h. A medium containing at least 2% ofrose can be 40 inoculated with a preculture of a strain of Leuconostocenteroides ssp. cremoris according to the invention in 2 5 APR 2002er to allow the production of dextran sucrase in the culture medium, formple. A medium containing 5-12% of MSK medium (skimmed cow's 5 milk)plemented with 0.05-0.2% of yeast extract and at least 2% of sucrose,example, can be inoculated with 0.2-3% of a preculture of a strain ofconostoc mesenteroides ssp. cremoris according to the present invention,ticularly with 0.2-3% of a preculture of the 10 strain CNCM I-1692 orstrain CNCM I-1693. It can be allowed to ferment at 25-35C for 10-20 hh the pH being maintained at 6-7.3, for example. A medium containing5-0.2% of yeast extract and at 15 least 2% of sucrose, for example, caninoculated with 0.2-3% of a preculture of a strain of Leuconostocenteroides ssp. cremoris according to the present invention,ticularly with 0.2-3% of a preculture of the strain CNCM I-1692 or theain CNCM I-1693. It can be 20 allowed to ferment at 25-35C for 10-20 hh the pH being maintained at 6-7.3, for example. When fermentation hased, this culture can be mixed with an equal volume of 20% MSK medium sot inhibition of the production of the enzyme dextran sucrase by thetose contained in the MSK 25 medium, for example, can be avoided duringfermentation. Also, a synthetic culture medium containing at least 2%sucrose, 1-3% of K2HP04, 0.2-1% of yeast extract, 0.2-1% of 30 peptone0.0005-0.001% of MnS04 can be inoculated with 0.2-3% of a preculture oftrain of Leuconostoc mesenteroides ssp. cremoris according to thesent invention, particularly with 0.2-3% of a preculture of the strainM I-1692 or the strain CNCM I-1693. It can be 35 allowed to ferment at35C for 7-12 h with the pH being maintained at 6-7.3, for example. In ast preferred embodiment of the process for the preparation of anitive containing active dextran 40 sucrase, the pH of the culture isusted to 5-5.5 after fermentation and this culture is then dried to give2 5 APR 2002 s powder containing the active dextran sucrase. The pH ofculture can be adjusted by the addition of lactic acid, for example.culture can be dried by lyophilization or by spray drying, for example.n a second preferred embodiment of the process for the preparation ofive dextran sucrase, the culture is separated after fermentation so asisolate the supernatant containing the active dextran sucrase. Thisn precipitate the macromolecules contained in this supernatant so as tolate a precipitate containing the dextran sucrase, for example. Theromolecules in the supernatant can be precipitated with polyethylenecol or ammonium sulphate at 2-6C, with stirring, for example. 20 Thecipitate containing the dextran sucrase can then be dialyzed so as tominate the precipitating agents, for example. 25 Finally, in this secondferred embodiment of the present process, the precipitate containing thetran sucrase can be stored at a temperature below -4C after thecipitation step or after the dialysis step, for example. 30 The presentention further relates to a food product or cosmetic compositionprising an additive containing active dextran sucrase from Leuconostocenteroides ssp. cremoris obtainable by carrying out the above process.To prepare such a product or composition, an additive containing activetran sucrase obtained in this way can be incorporated into a food ormetic product, such as a milk powder, a yoghurt, a ketchup, a mayonnaisea skin 40 cream, during its manufacture, for example. 2 5 APR 2002 9ally, the present invention further relates to a method of using aain of Leuconostoc mesenteroides ssp. cremoris according to the presentention for the manufacture of a food product or cosmetic composition. 5strains of Leuconostoc mesenteroides ssp. cremoris, the dextran sucraseduced by these strains and the dextran synthesized by these strainsording to the present invention are characterized in greater detail 10ow by means of different microbiological and biochemical dataustrating their properties. The percentages are given by weight, unlessicated otherwise. 15 Testing for the strains of Leuconostoc whichduce dextran Tests for the strains which produce dextran were performed150 strains isolated either from dairy products or from 20 non-dairyducts such as wine, coffee and sauerkraut. The production of dextran inedium containing sucrose is measured. 25 To do this, 10 ml of DEX mediumprising 1% of B. tryptone, 0.5% of yeast extract, 0.5% of K2HP04, 0.5%ammonium citrate and 5% of sucrose are inoculated with 1% of aculture of each of the 150 strains. The medium is then allowed toment at 30C for 24 h. 30 16 strains capable of producing dextran weres selected from the 150 starting strains. 150 ml of DEX medium, ascribed above, are then 35 inoculated with 1% of a preculture of each ofse 16 selected strains and then allowed to ferment at 30C for 24 h priormeasurement of the viscosity of the product of these 16 culturesained in this way. The viscosity is measured with a gravity viscometerdiameter 25 mm. 40said Table, the strains A are strains of Leuconostoc sp., the strains Bstrains of Leuconostoc mesenteroides ssp. cremoris, the strain C1 is aain of Leuconostoc lactic and the strains D are strains of 10conostoc mesenteroides spp. mesenteroides. Table I StrainViscosityA111A216A314A413A518A620A719A819C117CNCM69222B112B214B315D112D214D315D417 15 The results shown in Table I aboveonstrate the fact that the strain Leuconostoc meseateroides ssp.moris CNCM I-1692 has the highest viscosity of the 16 selected strains,it is acknowledged that the strain Leuconostoc 20 mesenteroides ssp.moris CNCM I-1692 produces the greatest amount of dextran. 2 5 APR2 m Study of the concentration of the enzyme dextran sucrase as action of fermentation time A synthetic medium containing 2% of sucrose,of K2HP04, 5 0.5% of yeast extract, 0.5% of peptone, 0.02% of MgS04,01% of MnS04, 0.001% of FeS04 and 0.001% of NaCl is inoculated with 1%a preculture of the strain Leucvnostoc mesenteroides ssp. cremoris CNCM692. It is allowed to ferment at 30C for 12 h in a 5 1 fermenter. 10er 4 h of fermentation, a sample of the resulting culture is taken everyours, up to a fermentation time of 12 h, for measurement of the growththe strain by means of the optical density at 600 nm. 15 Each sample isn centrifuged at 18,000 g for 20 min at 4C, the supernatant isovered, its pH is adjusted to 5.2 and the activity of the dextranrase contained in the supernatant is verified by measurement of the 20orporation of radioactivity into the dextran from radioactive sucroseDent. Res. 1974, 53, 1355-1360). Table II below shows the resultsained for measurement of the growth of the strain on the basis ofples taken 25 every 2 hours between fermentation times of 4 and 12 h.le II also shows the results of measurement of the activity of thetran sucrase contained in the supernatant of these samples. 30 Table IImentation time (h)4681012growth (ODsoo) / 100 . 0340 . 0850.210.510activity (u / ml)-0.10.l70.60.61 The results given in Table II demonstratefact that 35 the concentration of dextran sucrase reaches its maximumtaining 0.2% of sucrose, 0.5% of yeast extract, 0.5% of peptone, 2% ofP04, 0.02% of MgS04, 0.001% of MnS04, 0.001% of FeS04 and 0.001% of NaClinoculated with 1% of a preculture of Leuconostoc mesenteroides ssp.moris CNCM I-1692 and allowed to 10 ferment at room temperature for 12 hh the pH being maintained at a value of 6.7. The culture prepared ins way is then centrifuged at 18,000 g for 20 min at 4C. 15 Theernatant containing the dextran sucrase is then isolated and its pH isusted to a value of 5.2 prior to incubation for 4 h at 4C. 20 Theernatant is then mixed with an equal volume of 33% polyethylene glycoland the mixture is allowed to incubate at 4C for 5 h, with stirring, soto precipitate the proteins contained in the supernatant. 25 The mixturecentrifuged at 18,000 g for 20 min at 4C so as to isolate the residuetaining the precipitated proteins. This residue containing thecipitated proteins is then 30 suspended in 70 ml of 20 mM ammoniumtate, pH 5.2. 600 ~.g of dextranase are then added to this suspensionthe whole is allowed to incubate at 25C for 1 h so that the dextrantained in the suspension is digested by the 35 dextranase. Thepension is dialyzed in the presence of 70 ml of 20 mM ammonium acetate,5.2, so as to eliminate the glucose molecules obtained after digestionthe dextran 40 with the dextranase. The proteins are then isolated on anon exchange column (Fast Q, Pharmacia Biotech AB, 2 5 APR 2002 13sala, SU) which has been equilibrated beforehand with a 20 mM ammoniumtate buffer, pH 5.2. The proteins are eluted over a linear gradient of.5 M 5 NaCl. The activity of the dextran sucrase in the differenttein fractions eluted in this way is analyzed and these proteinctions are subjected to electrophoresis on an 10 SDS-polyacrylamide gel.protein fractions containing the dextran sucrase are isolated and theivity of the purified dextran sucrase is measured and found to be 105g.onstrated, on the one hand at 30C and on the other hand at 4C, therebyifying the texturing capacity of the dextrans at these differentperatures. 25 This is done by preparing an additive containing activetran sucrase from a culture of the strain Leuconostoc mesenteroides ssp.moris CNCM I-1692. A synthetic medium containing 2% of sucrose, 2% ofP04, 30 0.5% of yeast extract, 0.5% of peptone, 0.02% of MgS04, 0.001%MnS04, 0.001% of FeS04 and 0.001% of NaCl is inoculated with 1% of aculture of the strain Leuconostoc mesenteroides ssp. cremoris CNCM692. It is allowed to ferment at 30C for 12 h with the pH being 35ntained at 6.7. The culture is then separated by centrifugation at000 g for 20 min at 4C so as to isolate the supernatant containing thetran sucrase. 40 2 5 APR ~Q!!~ 14 The pH of the supernatant is lowered5.2 and this supernatant is incubated for 12 h at 4C. The macromoleculesthe supernatant are then 5 precipitated twice with polyethylene glycol4C so as to purify the dextran sucrase. The texturing capacity of thetrans synthesized by the dextran sucrase isolated in this way is thenified by 10 incubating the latter, on the one hand at 30C and on theer hand at 4C, in a buffer-substrate containing 20 mM acetate, pH 5.2,mM sucrose and 20 mM CaCl2. Table IV below shows the results ofturing with the 15 active dextran sucrase from the strain Leuconostocenteroides ssp. cremoris CNCM I-1692, on the one hand at 3 0C and on theer hand at 4C . Table IV 20 mediumincubation conditionstexturinga30C forh-4C for 12 h+++++ a: buffer-substrate medium of pH 5.2 . absence ofture +++++: good texture, very thick 25 The results shown in Table IVonstrate the fact thatobtained. This difference in texturing is without doubt due to the factt at 4C the dextran is in the form of molecules with short branches ands makes it possible to obtain a thick and pleasant texture, whereas atthe dextran is in the form of molecules 35 with long branches alignedallel with the main chain. These molecules do not affo'r~d a goodture. 2 5 APR 2002 15 Texturiag of a dairy product With the dextranrase The dextran sucrase is incubated under conditions (medium,perature and pH) identical to those which prevail 5 during theparation of a yoghurt. This is done by preparing an additive containingive dextran sucrase from a culture of the strain Leuconostocenteroides ssp. cremoris CNCM I-1692. 10 A synthetic medium containingof sucrose, 2% of K2HP04, 0.5% of yeast extract, 0.5% of peptone, 0.02%MgS04, 0.001% of MnS04, 0.001% of FeS04 and 0.001% of NaCl is inoculatedh 1% of a preculture of the strain 15 Leuconostoc mesenteroides ssp.moris CNCM I-1692. It is allowed to ferment at 30C overnight with the pHng maintained at 6.7. The culture is then separated by centrifugation at18,000 g for 20 min at 4C so as to isolate the supernatant containingdextran sucrase. The pH of the supernatant is then lowered to 5.2 ands supernatant is incubated for 12 h at 4C. 25 The macromolecules in theernatant are then precipitated twice with polyethylene glycol at 4C soto purify the dextran sucrase. 30 The synthesis of dextran by thetran sucrase is then verified. This is done by incubating the dextranrase on the one hand in a buffer-substrate of pH 6.4 containing 20 mMtate, 200 mM sucrose and 20 mM CaCl2, and on the other hand in a milknk containing 6% of sucrose. 35 Table V below shows the results obtainedtexturing with the dextran sucrase from the strain Leuconostocenteroides ssp. cremoris CNCM I-1692. 2 5 APR 2002 164C and incubation for 24 h at 4 C d: 5 h at 20C followed by adjustmentthe pH to 4.7, lowering of the temperature to 4C and incubation for 24 hat 4C . absence of texture +: small amount of texture +++++: goodture 15 The results shown in Table V make it possible to demonstrate thet that the dextran sucrase also textures a milk-based medium, but noger produces thickening dextran after 5 h at 37C. Yoghurts can betured by adding the dextran sucrase after fermentation 20 and beforerage at 4C. The Examples below are given in order to illustrate the usethe dextran, the dextran sucrase and / or a strain producing this dextranthis dextran sucrase in the 25 manufacture of a food product ormetic composition according to the present invention. The percentagesen are by weight, unless indicated otherwise. 30 Example 1 The strainconostoc mesenteroides ssp. cremoris CNCM I1692 according to the presentention is used for the manufacture of yoghurts. 35 . . ~ 2 5 APR 2002To do this, 1 1 of a milk product containing 2.8% of fats andplemented with 2% of skimmed milk powder and 6% of sucrose is prepared,is pasteurized at 96C for 30 min and its temperature is then lowered to. 5 In a parallel operation, a frozen preculture of a nonthickeningain of Streptococcus thermophilus and a frozen preculture of a-viscous strain Lactobacillus bulgaricus are reactivated in a sterileculture medium 10 containing 10% of reconstituted milk powder and 0.1%commercial yeast extract. A frozen preculture of the strain ofconostoc mesenteroides ssp. cremoris is also reactivated in an MRS 15ture medium (MRS lactobacilli - Detroit - USA) and then in a sterile MSKture medium containing 10% of reconstituted milk powder and 0.1% ofmercial yeast extract and supplemented with 1% of sucrose. 20 Theteurized milk product is then inoculated with 1% of each of thesectivated precultures and this milk product is then allowed to ferment atuntil the pH reaches a value of 4.5.s at 30 4C. Example 2 35 The additive containing active dextran sucraseording to the present invention is used for the manufacture of yoghurts.do this, a synthetic medium containing 2% of sucrose, 40 2% of KZHP04,% of yeast extract, 0.5% of peptone, 0.02% of MgS04, 0.001% of MnS04,01% of FeS04 and 0.001% of 2 5 APR 2002 18 NaCl is inoculated with 1%a preculture of the strain Leuconostoc mesenteroides ssp. crernoris CNCM692. It is allowed to ferment at 30C for 12 h with the pH beingntained at 6.7. The culture is then separated by 5 centrifugation at000 g for 20 min at 4C so as to isolate the supernatant containing theive dextran sucrase. The pH of the supernatant is lowered to 5.2 and theromolecules in the supernatant are then precipitated at 4C with ammoniumphate so as to isolate 10 a precipitate containing the dextran sucrase.s precipitate is then dialyzed to remove the ammonium sulphate. 1 1 of ak product containing 2.8% of fats and 15 supplemented with 2% of skimmedk powder and 6% of sucrose is also prepared, it is pasteurized at 96C30 min and its temperature is then lowered to 42C. In a parallelration, a frozen preculture of a non20 thickening strain ofeptococcus thermophilus and a frozen preculture of a non-viscous strainLactobacillus bulgaricus are reactivated in a sterile MSK culture mediumtaining 10% of reconstituted milk powder and 0.1% of commercial yeastract. 25 The pasteurized milk product is inoculated with 1% of each ofcultures of the two strains and the milk product is then incubated atuntil the pH reaches a value of 4.5. 30 1~ of purified dextran sucrasethen added, with stirring. Yoghurts are produced in this way and stored4C. 35 These yoghurts, prepared with the purified dextran sucrasethesized by Leuconostoc mesenteroides ssp. cremoris, have an unctuousture with a pleasant taste, especially after storage for 10 days at 4C.APR 2002 19 Example 3ract and 2% of sucrose is inoculated with 1% of a preculture of theain of 10 Leuconostoc mesenteroides ssp. cremoris. It is allowed toment at 30C for 20 h with the pH being maintained at 6.7. The culture ised with an equal volume of 20% MSK solution. The pH of the resultingture is then lowered to 5.2 by the addition of lactic acid before theture 15 is spray-dried to give the additive containing active dextranrase in powder form. 1 1 of a milk product containing 2.8% of fats andplemented with 2% of skimmed milk powder and 6% of 20 sucrose is alsopared, it is pasteurized at 96C for 30 min and its temperature is thenered to 42C. In a parallel operation, a frozen preculture of athickening strain of Streptococcus thermophilus and a 25 frozenculture of a non-viscous strain of Lactobacillus bulgaricus arectivated in a sterile MSK culture medium containing 10% of reconstitutedk powder and 0.1% of commercial yeast extract. 30 The pasteurized milkduct is inoculated with 1% of each of the cultures of the two strainsthe milk product is then incubated at 40C until the pH reaches a value4.5. 1% of the additive containing the active dextran sucrase is thened in powder form, with stirring. 35 Yoghurts are produced in this waystored at 4C. These yoghurts, prepared with the additive containingive dextran sucrase in powder form, have an unctuous 40 texture with aasant taste, especially after storage for 10 days at 4C. 2 5 APR 2002Example 4 The additive containing active dextran sucrase according 5 topresent invention is used in powder form for the manufacture of iceams. To do this, a culture medium containing 0.5% of yeast extract andleast 2% of sucrose is inoculated with 1% 10 of a preculture of theain of Leuconostoc mesenteroides ssp. cremoris. It is allowed to ferment23C for 20 h with the pH being maintained at 6.7. The pH of theulting culture is lowered to 5.2 by the addition of lactic acid. Theture is mixed with an equal volume of 15 20% MSK solution before beingay-dried to give the additive containing active dextran sucrase inder form. In another operation, 100 1 of an ice cream mixture containingof fats, 10% of non-fat solids, 14% of 20 sucrose, 3% of glucose syrup36-40, 0.3% of emulsifier, monoglyceride and diglyceride are prepared.s mixture has a total dry extract of 35.28%. The mixture prepared ins way is stirred at 60-65C for 20 min, homogenized at 210 bar and at 72Csing homogenization - 2 stages), 25 pasteurized at 86C for 22 sec andn cooled to +4C. The output of the homo-pasteurization line is 200 1 / h.mixture is acidified to pH 5.5 with lactic acid. 1% of the additivetaining the active dextran sucrase is 30 then added in powder form, withrring.ume increase, at a counterpressure of 3 bar and with an output of 80. The ice cream is then stored at -35C in a hardening chamber andsequently at -30C or -20C. 40 The ice cream prepared with the additivetaining active dextran sucrase in powder form has a good texturallity 2 5 APR 2002 21 and a pleasant taste. The ice cream obtained ins way gives a good sensation in the mouth. The ice cream is smooth andtuous. After accelerated ageing the ice cream preserves a good level oftural quality and of 5 creamy sensation in the mouth. The smoothracter of the ice cream is preserved well; this can be explained by theoprotective property of the dextrans, which then limits excessive growththe ice crystals. 10 Example 5 The additive containing active dextranrase according to the present invention is used in powder form for theufacture of ice creams. 15 To do this, a culture medium containing 0.5%yeast extract and at least 2% of sucrose is inoculated with 1% of aculture of the strain of Leuconostoc mesenteroides ssp. cremoris. It isowed to ferment at 23C for 20 h 20 with the pH being maintained at 6.7.pH of the resulting culture is lowered to 5.2 by the addition of lacticd. The culture is mixed with an equal volume of 20% MSK solution beforeng spray-dried to give the additive containing active dextran sucrase inder form. 25 In another operation, 100 1 of a premix containing 18% ofrose are prepared. 1% of the additive containing the active dextranrase is then added in powder form, with stirring. The premix is thenubated at +4C. 30 After incubation, the viscosity of the premix hasreased. The premix is made up to give a final ice cream mixturetaining 8% of fats, 10% of non-fat solids, 14% of sucrose, 3% of glucoseup DE 36-40, 0.3% 35 of emulsifier, monoglyceride and diglyceride. Thisture has a total dry extract of 35.28%. The mixture prepared in this waystirred at 60-65C for 20 min, homogenized at 210 bar and at 72C (risingogenization 2 stages), pasteurized at 8'6C for 22 sec and then cooled 40+4C. At this point the enzyme is totally inactivated. The output of theo-pasteurization line is 200 1 / h. 2 5 APR 2002 22 The mixture isured at +4C and then frozen on a freezer at -5C, with a 95% volumerease, at a counterpressure of 3 bar and with an output of 80 1 / h.m 10 has a good textural quality and a pleasant taste. The ice creamained in this way gives a good sensation in the mouth. The ice cream isoth and unctuous. After accelerated ageing the ice cream preserves ad level of textural quality and of creamy sensation in the mouth. 15 Theoth character of the ice cream is preserved well; this can be explainedthe cryoprotective property of the dextrans, which then limits excessivewth of the ice crystals. 2 5 APR 2002 23 Claims 1. Strain ofconostoc mesenteroides ssp. cremoris 5 which produces dextran. 2. Strainording to Claim 1 deposited in the CNCM under the number I-1692. 10 3.ain according to Claim 1 deposited in the CNCM under the number I-1693.Strain according to Claim 1 which produces the same dextran as theain CNCM I-1692 or the strain CNCM I15 1693. 5. Strain according to oneClaims 1, 3 and 4 which does not ferment lactose. 20 6. Process for theduction of dextran, wherein: - a culture medium containing sucrose isculated with a preculture of a strain according to one of Claims 1-5, -is allowed to ferment at 25-35C for 10-20 h, and - the pF of theulting culture is then lowered to 5-5.5 25 prior to storage at 0-10C for48 h. 7. Process according to Claim 6 wherein the culture mediumtains at least 20 of sucrose. 30 8. Process according to Claim 6 whereinculture is mixed with a milk-based medium just after the fermentationp. 9. Process according to Claim 6 wherein the culture is 35 dried afterstorage step. 10. Food product or cosmetic composition comprisingtran from Leuconostoc mesenteroides ssp. cremoris obtainable by carryingthe process according to one of 40 Claims 6-9. 2 5 APR 2002 24 11.cess for the production of an additive containing active dextranrase, wherein a medium containing sucrose is inoculated with aculture of a strain according to one of Claims 1-5 and is then alloweder fermentation and this culture is then dried. 14. Process according toim 11 wherein the culture is 15 separated after fermentation so as tolate the supernatant containing the dextran sucrase. 15. Processording to Claim 14 wherein the pH of the supernatant is adjusted to-5.7, the supernatant is 20 stored at 0-10C for 15-30 h and theromolecules contained in the supernatant are then precipitated so as tolate a precipitate containing the dextran sucrase. 16. Process accordingClaim 15 wherein the precipitate 25 containing the dextran sucrase islyzed. 17. Process according to one of Claims 15-16 wherein thecipitate containing the dextran sucrase is stored at a temperature below. 30 18. Food product or cosmetic composition comprising an additivetaining active dextran sucrase from Leuconostoc mesenteroides ssp.moris obtainable by carrying out the process according to one of Claimsto 35 16. 19. 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Claims

1. Souche de Leuconostoc mesenteroides ssp. cremoris produisant du dextrane.

2. Souche selon la revendication 1, déposée à la CNCM sous le numéro I-1692.

3. Souche selon la revendication 1, déposée à la CNCM sous le numéro I-1693.

4. Souche selon la revendication 1, produisant le même dextrane que la souche CNCM I-1692 ou que la souche CNCM I-1693.

5. Souche selon l'une des revendications 1,3 et 4, ne fermentant pas le lactose.

6. Procédé de production de dextrane, dans lequel:on inocule avec une préculture d'une souche selon l'une des revendications 1-5 un milieu de culture contenant du saccharose,on laisse fermenter à 25-35 DEG C pendant 10-20 h,puis on abaisse le pH de la culture ainsi obtenue à 5-5,5, avant de la stocker à 0-10 DEG C pendant 16-48 h.

7. Procédé selon la revendication 6, dans lequel le milieu de culture contient au moins 2% de saccharose.

8. Procédé selon la revendication 6, dans lequel on mélange la culture à un milieu laitier juste après l'étape de fermentation.

8. Procédé selon la revendication 6, dans lequel on sèche la culture après l'étape de stockage.

9. Composition alimentaire ou cosmétique, comprenant du dextrane de Leuconostoc mesenteroides ssp. cremoris susceptible d'être obtenu par la mise en oeuvre du procédé selon l'une des revendications 6-8.

10. Procédé de production d'un additif contenant de la dextrane-sucrase active dans lequel on inocule avec une préculture d'une souche selon l'une des revendications 1-5 un milieu contenant du saccharose, puis on laisse fermenter à 25-35 DEG C pendant 10-20 h.

11. Procédé selon la revendication 10, dans lequel le milieu de culture contient au moins 2% de saccharose.

12. Procédé selon la revendication 10, dans lequel, après la fermentation, on ajuste le pH de la culture à 5-5,5 puis on sèche cette culture.

13. Procédé selon la revendication 10, dans lequel on réalise une séparation sur la culture après la fermentation, de manière à isoler le surnageant contenant la dextrane-sucrase.

14. Procédé selon la revendication 13, dans lequel on ajuste le pH du surnageant à 4,9-5,7, on le stocke à 0-10 DEG C pendant 15-30 h, puis on précipite les macromolécules contenues dans le surnageant, de manière à isoler un précipité contenant la dextrane-sucrase.

15. Procédé selon la revendication 14, dans lequel on effectue une dialyse du précipité contenant la dextrane-sucrase.

16. Procédé selon l'une des revendications 14-15, dans lequel on stocke le précipité contenant la dextrane-sucrase à une température inférieure à -4 DEG C.

17. Composition alimentaire ou cosmétique, comprenant un additif contenant de la dextrane-sucrase active de Leuconostoc mesenteroides ssp. cremoris susceptible d'être obtenu par la mise en oeuvre du procédé selon l'une des revendications 10 à 16.

18. Procédé d'utilisation d'une souche selon l'une des revendications 1-5, pour la fabrication d'une composition alimentaire ou cosmétique.